Methods and compositions for producing hepatocyte-like cells

ABSTRACT

Methods are provided for producing a population of hepatocyte-like cells (iHeps) from a population of adipocyte-derived stem cells (ASCs). Aspects of the methods include placing a population of ASCs into a three dimensional culture (e.g., hanging drop suspension culture, high density culture, spinner flask culture, microcarrier culture, etc.), and contacting the cells with a first and second culture medium. Also provided are methods of treating an individual, which include producing a population of iHeps from a population of ASCs, and administering an effective number of iHeps into the individual. Kits for practicing the methods are also described herein.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 14/917,558 filed Mar. 8, 2016, which is a national stage entryof PCT Application No. PCT/US2014/056049, filed Sep. 17, 2014, whichclaims benefit of U.S. Provisional Patent Application No. 61/880,056,filed Sep. 19, 2013, which applications are incorporated herein byreference in their entirety.

GOVERNMENT RIGHTS

This invention was made with Government support under contract DK090992awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND

A major goal for regenerative medicine is to facilitate human tissuereplacement through transplantation of stem cells that can be harvestedfrom readily accessible tissues. For example, orthotopic livertransplantation is the only effective treatment for end-stage liverdisease or severe liver injury, but its utility is severely limited bythe lack of donor liver tissue and by the requirement for lifelongimmunosuppression.

A large number of adipocyte-derived stem cells (ASCs) can be easilyobtained using a commonly performed procedure, liposuction.Additionally, methods for inducing ASC differentiation intohepatocyte-like cells (iHeps) have been developed. Liver regenerationvia transplantation of iHeps (e.g., autologous iHeps) is a highlyattractive possibility for regenerative medicine. By this method, ASCobtained by liposuction are induced to differentiate into iHeps invitro, and then iHeps are transplanted into the donor's liver. Theabundance and accessibility of adipose tissue ensures that there is asource of readily available ASCs. Moreover, liver regeneration usingautologous iHeps would not require immunosuppression.

However, since a patient can die rapidly after acute liver failure(e.g., caused by acetaminophen toxicity), the prolonged culture periodassociated with the currently known method to produce iHeps from ASCsseverely limits the clinical utility for producing iHeps. Furthermore,the known method to produce iHeps from ASCs is also characterized by lowefficiency and low yield.

The production of hepatocyte-like cells from adipocyte-derived stemcells for use in therapy and/or research will benefit from reduced cost,reduced culture time, increased efficiency, and increased yield.

PUBLICATIONS

-   Amos et al., Tissue Eng Part A. 2010 May; 16(5):1595-606; Maclsaac    et al., Exp Cell Res. 2012 Feb. 15; 318(4): 416-423; Kapur et al,    Biofabrication. 2012 Jun.; 4(2):025004; Gutierrez et al., Exp    Hematol. 2005 October; 33(10):1083-91; Banerjee et al,    Cytotechnology. 2006 May; 51(1):1-5. -2006; Banas et al.,    Hepatology. 2007 July; 46(1):219-28; Banas et al., J Gastroenterol    Hepatol. 2009 Jan.; 24(1):70-7; Green et al., 2011. Nat Biotechnol    29:267-272; So et al., Biol Open. 2013 Jan. 15; 2(1):30-6; Payne et    al., Vitam Horm. 2011; 85:207-16; Hay et al., Proc Natl Acad Sci    USA. 2008 Aug. 26; 105(34):12301-6; Lue et al, Liver Int. 2010 Jul.;    30(6):913-22; Hasegawa et al, Biochem Biophys Res Commun. 2011 Feb.    18; 405(3):405-10; Haraguchi et al, J Tissue Eng Regen Med. 2013    Jun. 3; Cameron et al, Biotechnol Bioeng. 2006 Aug. 5; 94(5):938-48;    WO2007089798; US20100098739; and US20100112031.

SUMMARY

Methods are provided for producing a population of hepatocyte-like cells(iHeps) from a population of adipocyte-derived stem cells (ASCs).Aspects of the methods include placing a population of ASCs into a threedimensional culture (e.g., hanging drop suspension culture, high densityculture, spinner flask culture, microcarrier culture, etc.) andcontacting the cells with a first and second culture medium. Alsoprovided are methods of treating an individual, which include producinga population of iHeps from a population of ASCs, and administering aneffective number of iHeps into the individual.

The presence of iHeps may be confirmed by various methods, including forexample, contacting the induced cell population with an antibodyspecific for a hepatocyte marker protein, and determining the percentageof cells positive for expression. In some embodiments, 15% or more ofcells of the induced cell population are hepatocyte-like cells. In somecases, 37% or more of the cells of the induced cell population arehepatocyte-like cells. In some embodiments, the elapsed time to generateiHeps from ASCs is less than 13 days (e.g., less than 10 days). The highefficiency of iHep production and the short time frame over which iHepscan be produced by the subject methods facilitate treatment methodsbecause liver damage (e.g., liver failure, e.g., due to acetaminophentoxicity) can rapidly cause death (e.g., in 2 weeks or less). Kits forpracticing the methods of the disclosure are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIGS. 1A-1E provide a comparison of SCi-Heps to Chi-Heps, and acomparison of the methods of generating SCi-Heps and Chi-Heps. (FIG. 1A)The two different methods for inducing the differentiation of ASC intoiHeps are shown. The top panel shows the method for chemicallydifferentiating ASC into Chi-Heps. The cells isolated fromlipo-aspirates are cultured for 3-15 days ASC cells. These cells arethen differentiated from mesoderm into endodermal cells over a 3-dayperiod (stage 1), and then further differentiated into Chi-Heps (stage2) using defined media containing growth factors. The bottom panel showsone embodiment of a subject method for producing SCi-Heps. (FIG. 1B)Bright field images (100×) showing the change in the morphology of thespindle-shaped ASC as they differentiate into Chi-Heps (via chemicaldifferentiation) or SCi-Heps using a subject method for producingSCi-Heps. Relative to Chi-Heps, SCi-Heps have a greater cellular densityand colony-like morphology that more closely resembles hepatocytes.(FIG. 1C) The percentage of ASC, Chi-Heps, SCi-Heps and iPS-Hepsexpressing the CK8/18 marker found on mature hepatocytes. Unstained ASCwere used to characterize the background level of staining (control),and 100% of human hepatocytes express this marker. (FIG. 1D)Immunofluorescence staining images (at 100×) of control ASC, Chi-Heps,SCi-Heps or hepatocytes for human CK8/18 expression, LDL uptake or PASstaining. Chi-Heps and SCi-Heps, but not ASC, can endocytose LDL and cansynthesize glycogen (PAS stain). (FIG. 1E) The amount of human albuminor urea secreted into the supernatant by Chi-Heps, SCi-Heps and ASCcultured for the indicated time period. SCi-Heps produced albumin andurea well before Chi-Heps produced detectable amounts of these analytes,while ASC did not produce albumin or urea. In panels C and E, each barrepresents the average (+SEM) of 4 biologically independent samplesanalyzed.

FIGS. 2A-2C depict gene expression in Chi-Heps, SCi-Heps and adipocytes.(FIG. 2A) iHeps have increased levels of hepatocyte-specific mRNAs anddecreased levels of adipocyte-specific mRNAs. RT-PCR analysis was usedto measure the level of hepatocyte-specific (FoxA2, AFP, ALB, AAT, TDO2)or adipocyte-specific (CD37) mRNA expression in Chi-Heps, SCi-Heps,iPS-Heps or ASCs. For each gene, the mRNA levels in Chi-Heps (after 15days of differentiation), SCi-Heps (after 9 days of differentiation) andiPS-Heps (after 30 days of differentiation) are normalized relative tothat in ASCs. (FIG. 2B) RT-PCR analysis of the gene expression in ASCs,and in SCi-Heps after 3, 6 and 12 days of induced differentiation. Thelevel of CD105 mRNA expression (which is an ASC marker) was decreased by35-fold (p=0.001); while the level of hepatocyte (FoxA2 and ALB) mRNAexpression was increased by 25- and 51-fold, respectively (p=0.0007 and9×10⁻⁵, respectively), in SCi-Heps after 12 days of hepatocytedifferentiation. The mRNA levels in SCi-Heps were normalized relative tothat in ASCs. Each data point in panels A-B represents the average±SEMfor 3 independent determinations. As shown in FIG. 11, there weresignificant changes in the level of expression of each gene in SCi-Hepsafter 3, 6 and 12 days of differentiation. (FIG. 2C) An illustration ofthe spatial relationship between the gene expression profiles obtainedfrom ASCs, Chi-Heps, SCi-Heps, iPS-Heps and hepatocytes.Microarray-based global gene expression data was analyzed as describedin the methods section. The lengths of each connecting edge (and thenumber shown) indicate the distance between the expression profiles forthe cell types at each of the corresponding vertices. This distance isdetermined by summing the squares of the differences in the level ofexpression for each gene on the array for the two cell types at thevertices of each line. This diagram indicates that the gene expressionpattern in Chi- and SCi-Heps is closer to that of hepatocytes than isthat of iPS-Heps. Also, the deviation of the iPS-Heps pattern from theASC-hepatocyte axis is larger than that of iHeps, which indicates thatiPS-Heps have a larger number of gene expression changes that areexternal to both ASCs and hepatocytes than are found in Chi- orSCi-Heps.

FIGS. 3A-3D demonstrate the implantation (via injection) of SCi-Hepsinto the mouse liver. (FIG. 3A) An ultrasound generated image (intransverse view) of SCi-Heps being injected through a 30G needledirectly into the right lobe of the liver of a TK-NOG mouse. (FIG. 3B)Chimeric mice produced human albumin after SCi-Hep transplantation. Theamount of human albumin in plasma was serially measured over an 8-weekperiod after transplantation of SCi-Heps into 4 ganciclovir-conditionedmice. Each dashed line shows the amount of human albumin measured in achimeric mouse at the indicated time. In all 4 chimeric mice, humanserum albumin was detectable 4 weeks after transplantation, and theamount increased with time after transplantation. In contrast, albuminwas not produced by any of the 4 mice that were recipients of control,undifferentiated ASCs (red triangles and solid line). (FIG. 3C)Immunofluorescent staining of liver sections (at 100× magnification)obtained from TK-NOG mice 4 weeks after implantation of SCi-Heps, (a-c)or undifferentiated ASCs (d-f). The liver sections were stained withhuman specific anti-albumin (a, d), anti-CK8/18 (b, e), or anti ASGR1antibodies, and counter-strained with DAPI to show the cell nuclei.(FIG. 3D) Immunofluorescent staining of liver sections (200×magnification) obtained from TK-NOG mice 4 weeks after transplantationwith SCi-Heps or undifferentiated ASCs. The liver sections were stainedwith anti-CK8/18, anti-Ki67 or anti-ZO-1 antibodies; and were thencounter-strained with DAPI to show the cell nuclei.

FIGS. 4A-4B demonstrate that SCi-Heps do not form tumors inimmunocompromised mice, but iPS-Heps do. (FIG. 4A) As early as threeweeks after 5×10⁴ iPS-Heps were implanted under the kidney capsule ofNOG mice, palpable tumors were formed in the area of implantation. Incontrast, no tumors were detected 2-months after the same number of Chi-or SCi-Heps were implanted (top row). The tumors were visible in tissuesections obtained from the area of iPS-Hep implantation, while onlynormal tissue was present in the area of Chi-Hep implantation (middlerow). The magnification of images in the top and middle rows are 10×.The images in the bottom rows were obtained from the boxed regions ofthe corresponding image and are shown at 200× magnification. (FIG. 4B)Tumor formation after iPS-Heps implantation. Top row: Images of thetumors formed 3 weeks after iPS-Hep cells were implanted under thekidney capsule. The 300× and 100× images were obtained from theindicated region (dashed box) of the corresponding 40× and 100× images.Bottom row: Two months after the iPS-iHep cells were implanted, thetumors contained cells from all three germ cell layers. These images areat 200× magnification. The scale bars shown are 50 μm.

FIGS. 5A-5C provide FACS analysis performed using an anti-Sox 17antibody, which recognizes to a marker for endodermal cells. ASC wereisolated from three different donors and induced to differentiate usingthe standard (Chi-Hep) or spherical culture (SCi-Heps) methods. After 4days of differentiation, FACS was performed using an anti-Sox 17antibody, which recognizes to a marker for endodermal cells. Thepercentage of ASC differentiating into endodermal cells after sphericalculture (61.2±2.4) more than doubled that obtained using the standardmethod (25.7±1.3).

FIGS. 6A-6B provides FACS analysis performed using an anti-CD105antibody, which binds to a marker for ASC. ASC were isolated from threedifferent donors and induced to differentiate into SCi-Heps using thespherical culture method. After 9 days of differentiation, FACS wasperformed using an anti-CD105 antibody, which binds to a marker for ASC.While 98.6±0.5% of ASC expressed CD105, the percentage of SCi-Hepsexpressing this ASC marker was only 9.0±0.5%.

FIG. 7 demonstrates that iPS-Heps can synthesize glycogen (a,b) andendocytose low-density lipoproteins (LDL) (c,d). The LDLimmunofluorescence and PAS stained images are shown at 100× (a, c) and400× magnification (b, d). In contrast, ASC could not endocytose LDL (e,100×), nor did they stain with PAS (f, 100×).

FIGS. 8A-8D demonstrate that iPS-Heps have hepatocyte properties. Theamount of human albumin (FIG. 8A) or urea nitrogen (FIG. 8B) secretedinto the supernatant by Chi-Heps, iPS-Heps and ASCs are shown at theindicated time points. Chi-Heps produced albumin and urea well beforeiPS-Heps produced detectable amounts of these analytes, while controlASCs did not produce albumin or urea. (FIG. 8C) Chi-Heps (after 15 days)and iPS-Heps (after 30 days of differentiation), but not ASCs, canmediate a CYP3A4-dependent drug biotransformation reaction. (FIG. 8D)iPS-Heps have increased levels of hepatocyte-specific and decreasedlevels of adipocyte-specific mRNAs. RT-PCR analysis was used to measurethe level of hepatocyte-specific (FoxA2, AFP, ALB, AAT, A1AT, TDO2) oradipocyte-specific (CD37, CD29) mRNA expression in s, iPS-Heps or ASCs.For each gene, the mRNA levels in in Chi-Heps (after 15 days ofdifferentiation) and iPS-Heps (after 30 days of differentiation) arenormalized relative to that of the corresponding mRNA level in ASCs.Each bar or data point in panels A-D represents the average (±SEM) of 4biologically independent samples that were analyzed.

FIG. 9 provides principal component analysis for the gene expressiondata obtained from ASC, Chi-Hep, SCi-Hep, iPS-Hep and hepatocytes. Thefirst (x-axis) and the second (y-axis) principle components (PC)explained 46.4% and 26.5%, respectively, of the total variance in theexpression profile. Of note, iPS-Heps had a large amount of deviationalong PC2 relative to ASCs and hepatocytes, while Chi-Heps and SCi-hepswere right in between the ASCs and hepatocytes. Consistent with otheranalyses (including the analysis shown in FIG. 2C), this graph indicatesthat iPS-Heps had a large number of gene expression changes that werenot present in hepatocytes (relative to ASCs).

FIG. 10 demonstrates that SCi-Heps do not express ASGR-1 in vitro.Permeabilized human hepatocytes (a-c) or SCi-Heps (d-f), which wereanalyzed after 9 days of in vitro differentiation, were stained with ananti-human ASGR-1 antibody and counter-stained with DAPI. Theimmunofluorescence images (200× magnification) show that humanhepatocytes, but not SCi-Heps expressed ASGR-1. The scale bars shown are50 μm.

FIG. 11 presents a table depicting the numbers of differentiallyexpressed genes in hepatocytes, Chi-Heps, SCi-Heps, and iPS-hepsrelative to ASCs. From analysis of microarray data, the indicated numberof genes whose expression was significantly increased, decreased orunchanged in hepatocytes relative to ASCs were selected. (A gene'sexpression was considered as altered in hepatocytes if the absolutevalue of the fold change was >5 and the adjusted p value was <0.01.)Each of the 3 panels indicates the number (and percentage) of theselected genes whose expression was increased, decreased or notsignificantly changed (NS) when the expression profiles in Chi-Heps,SCi-Heps or iPS-Heps (relative to ASCs). For each of these comparisons,the expression of a gene was considered altered in iHeps if the absolutevalue of the fold change was >2 and the adjusted p value was <0.01. Itis noteworthy that 18% of the genes whose expression was not altered inhepatocytes had an altered level of expression in iPS-Heps.

FIG. 12 presents a table depicting changes in the level of expression ofgenes (Foxa2, Albumin) expressed in hepatocytes and inadipocyte-specific (CD105) mRNAs during induced SCi-Hep differentiation.RT-PCR was used to measure the level of expression of 3 genes (Foxa2,Albumin and CD105) in ASC, and in SCi-Heps after 3, 6 and 12 days ofinduced differentiation. The measured average expression levels for eachgene in SCi-Heps on the indicated day (relative to its level ofexpression in ASC) and the corresponding p-values for the expressiondifferences are shown. Each value is the average fold-change for 3independent measurements; a positive value indicates that its expressionwas increased in SCi-Heps relative to ASC, while a negative valueindicates that it was decreased. A one-sample t-test was applied usingthe log-transformed expression level to assess the statisticalsignificance (P-value) of the measured expression differences.

FIGS. 13A-13B demonstrate that ASCs cultured by stirred suspensionculture (spinner flask culture in this case) form a high density ofcellular aggregates that resemble the spheres formed during hanging dropsuspension culture. Images shown are at 10× magnification. (FIG. 13A)The morphology of ASC cellular aggregates formed after spinner flaskculture of ASCs for 24 hours. The morphology of the aggregates is verysimilar to that of the ‘spheres’ formed after culturing ASCs by thehanging drop method. The increased cell density (hence the term “highdensity culture”) in the spinner flask culture is readily apparent whencomparing panel (FIG. 13A) to panel (FIG. 13B). (FIG. 13B) ‘spheres’formed after culturing ASCs by the hanging drop method for 48 hours.

FIGS. 14A-14B demonstrate that ASCs cultured by stirred suspensionculture (spinner flask culture in this case) form a greater percentage(FIG. 14A) of CD34+ cells (adipose stem cells) and a roughly equalpercentage (FIG. 14B) of SOX17+ cells (endodermal precursor cells)compared to ASCs cultured by the hanging-drop method. X-axis is theintensity of signal detected.

FIGS. 15A-15B demonstrate that iHeps produced using spinner flaskculture (SS-Hep) had the specific cytochrome CYP enzymes CYP3A4 andCYP1A1, and the activity of CYP3A4 was strongly induced by dexamethasone(Dex) treatment. YJM is a human hepatocyte cell line. CYP activity wasnormalized to cell viability. Results are presented with (+) and without(−) Dex induction.

FIG. 16 demonstrates that iHeps produced using spinner flask culture(SS-Hep) secreted an increased level of human albumin (hAlb) compared toChi-Heps and SCi-Heps (approximately 16-fold relative to SCi-Heps andapproximately 96-fold relative to Chi-Heps).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods are provided for producing a population of hepatocyte-like cells(iHeps) from a population of adipocyte-derived stem cells (ASCs).Aspects of the methods include placing a population of ASCs into a threedimensional culture (e.g., hanging drop suspension culture, high densityculture, spinner flask culture, microcarrier culture, etc.), andcontacting the cells with a first and second culture medium. Alsoprovided are methods of treating an individual, which include producinga population of iHeps from a population of ASCs, and administering aneffective number of iHeps into the individual. Kits for practicing themethods are also described herein.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed

Definitions

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides). In some embodiments, theaffinity of one molecule for another molecule to which it specificallybinds is characterized by a K_(D) (dissociation constant) of 10⁻⁵ M orless (e.g., 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M orless, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³ M orless, 10⁻¹⁴ M or less, 10⁻¹⁵ M or less, or 10⁻¹⁶ M or less). “Affinity”refers to the strength of binding, increased binding affinity beingcorrelated with a lower K_(D).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member).

The term “specific binding agent” as used herein refers to any agentthat specifically binds a biomolecule (e.g., a marker such as a nucleicacid marker molecule, a protein marker molecule, etc.). In some cases, a“specific binding agent” for a marker molecule (e.g., a hepatocytemarker molecule) is used. Specific binding agents can be any type ofmolecule. In some cases, a specific binding agent is an antibody or afragment thereof. In some cases, a specific binding agent is nucleicacid probe (e.g., an RNA probe; a DNA probe; an RNA/DNA probe; amodified nucleic acid probe, e.g., a locked nucleic acid (LNA) probe, amorpholino probe, etc.; and the like)

As used herein, a “marker molecule” does not have to be definitive(i.e., the marker does not have to definitely mark the cell as being ofa particular type. For example, the expression of a marker molecule by acell can be indicative (i.e., suggestive) that the cell is of aparticular cell type. For example, if 3 cell types (type A, type B, andtype C) express a particular marker molecule (e.g., a particular mRNA, aparticular protein, etc.), expression of that marker molecule by a cellcannot necessarily be used by itself to definitively determine that thecell is a type A cell. However, expression of such a marker can suggestthat the cell is a type A cell. In some cases, expression of such amarker, combined with other evidence, can definitively show that thecell is a type A cell. As another illustrative example, if a particularcell type is known to express two or more particular marker molecules(e.g., mRNAs, proteins, a combination thereof, etc.) then the expressionby a cell of one of the two or more particular marker molecules can besuggestive, but not definitive, that the cell is of the particular typein question. In such a case, the marker is still considered a markermolecule.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibodies” (Abs) and“immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

As used in this disclosure, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

A “TK-NOG mouse”, as used herein refers to a mouse in which a herpessimplex virus type 1 thymidine kinase (HSVtk) transgene is expressedwithin the liver of a highly immunodeficient NOG mouse. Mouse livercells expressing the HSVtk transgene can be ablated after a briefexposure to a non-toxic dose of ganciclovir (GCV). In some embodiments,an individual receiving treatment can be a mouse. For example, a TK-NOGmouse with ablated liver cells can be considered an individual withreduced liver function (i.e., an individual with liver damage) and themouse can therefore be considered to be an individual receivingtreatment when subject iHEPS (describe in detail below) are transplantedinto the mouse. Transplanted human liver cells can be stably maintainedwithin the liver of TK-NOG mice, and TK-NOG mice with transplanted humanliver cells are referred herein as humanized TK-NOG mice. Thereconstituted liver of humanized TK-NOG mice can be a mature andfunctioning human organ, and can generate a human-specific profile ofdrug metabolism. The ‘humanized liver’ can be stably maintained inhumanized TK-NOG mice with a high level of function for a prolongedperiod (e.g., at least 8 months). For more information about TK-NOGmice, refer to: (i) Hasegawa et al, Biochem Biophys Res Commun. 2011Feb. 18; 405(3):405-10; (ii) Yamazaki et al, Chem Res Toxicol. 2012 Feb.20; 25(2):274-6; (iii) Hu et al., Pharmacogenet Genomics. 2013 Feb.;23(2):78-83; and (iv) Yamazaki et al, Chem Res Toxicol. 2013 Mar. 18;26(3):486-9; which are hereby incorporated by reference in theirentirety.

The term “stem cell” is used herein to refer to a mammalian cell thathas the ability both to self-renew and to generate a differentiated celltype (see Morrison et al. (1997) Cell 88:287-298). In the context ofcell ontogeny, the adjective “differentiated”, or “differentiating” is arelative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with. Thus, pluripotent stem cells can differentiate intolineage-restricted progenitor cells (e.g., adipocyte-derived stemcells), which in turn can differentiate into end-stage cells (e.g.,adipocytes, osteoblasts, chondrocytes, etc.), which play acharacteristic role in a certain tissue type, and may or may not retainthe capacity to proliferate further. Stem cells (and differentiatedprogeny) may be characterized by both the presence of specific markers(e.g., proteins, RNAs, etc.) and the absence of specific markers. Stemcells may also be identified by functional assays both in vitro and invivo, particularly assays relating to the ability of stem cells to giverise to multiple differentiated progeny.

The stem cells of interest are mammalian, where the term refers to cellsisolated from any animal classified as a mammal, including humans,domestic and farm animals, and zoo, laboratory, sports, or pet animals,such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In someembodiments, the mammal is a human and the mammalian cells (e.g., apopulation of adipocyte-derived stem cells) are therefore human cells(e.g., a population of human adipocyte-derived stem cells).

The terms “passaging” or “passage” (i.e., splitting or split) in thecontext of cell culture are known in the art and refer to thetransferring of a small number of cells into a new vessel. Cells can becultured if they are split regularly because it avoids the senescenceassociated with high cell density. For adherent cells, cells aredetached from the growth surface as part of the passaging protocol.Detachment is commonly performed with the enzyme trypsin and/or othercommercially available reagents (e.g., TrypLE, EDTA(Ethylenediaminetetraacetic acid), a policemen (e.g., a rubberpolicemen) for physically scrapping the cells from the surface, etc.). Asmall number of detached cells (e.g., as few as one cell) can then beused to seed a new cell population, e.g., after dilution with additionalmedia. Therefore, to passage a cell population means to dissociate atleast a portion of the cells of the cell population, dilute thedissociated cells, and to plate the diluted dissociated cells (i.e., toseed a new cell population).

The terms “media” and “medium” are herein used interchangeably. Cellculture media is the liquid mixture that baths cells during in vitroculture.

The term “population”, e.g., “cell population” or “population of cells”,as used herein means a grouping (i.e., a population) of two or morecells that are separated (i.e., isolated) from other cells and/or cellgroupings. For example, a 6-well culture dish can contain 6 cellpopulations, each population residing in an individual well. The cellsof a cell population can be, but need not be, clonal derivatives of oneanother. A cell population can be derived from one individual cell. Forexample, if individual cells are each placed in a single well of a6-well culture dish and each cell divides one time, then the dish willcontain 6 cell populations. A cell population can be any desired sizeand contain any number of cells greater than one cell. For example, acell population can be 2 or more, 10 or more, 100 or more, 1,000 ormore, 5,000 or more, 10⁴ or more, 10⁵ or more, 10⁶ or more, 10⁷ or more,10⁸ or more, 10⁹ or more, 10¹⁰ or more, 10¹¹ or more, 10¹² or more, 10¹³or more, 10¹⁴ or more, 10¹⁵ or more, 10¹⁶ or more, 10¹⁷ or more, 10¹⁸ ormore, 10¹⁹ or more, or 10²⁰ or more cells.

Methods

Aspects of the disclosure include methods of producing a population ofhepatocyte-like cells from a population of adipocyte-derived stem cells(ASCs). The methods generally involve placing a population of ASCs intoa three dimensional culture (e.g., hanging drop suspension culture, highdensity culture, spinner flask culture, microcarrier culture, etc.) toproduce an ASC-derived cellular aggregate (e.g., sphere); contactingcells of the ASC-derived cellular aggregate with a first culture mediumcomprising Activin A and a fibroblast growth factor (FGF) to produce aprecursor cell population; and contacting cells of the precursor cellpopulation with a second culture medium comprising hepatocyte growthfactor (HGF) to produce an induced cell population that comprisesinduced hepatocyte-like cells (iHeps).

Adipose derived stem cells (ASCs). The term “adipose-derived stem cell”refers to a population of adipose cells found in post-natal mammals thatare pluripotent and have the potential to differentiate into a varietyof cell types including but not limited to cells of osteogenic,adipogenic, and chondrogenic lineages. ASCs can also differentiate intohepatocyte-like cells. ASCs have been referred to in the literature asadipose-derived adult stem (ADAS) cells, adipose-derived adult stromalcells, adipose-derived stromal cells (ADSCs), adipose stromal cells(ASCs), adipose mesenchymal stem cells (AdMSCs), lipoblast, pericyte,preadipocyte, and processed lipoaspirate (PLA) cells. The InternationalFat Applied Technology Society (IFATS) reached a consensus to adopt theterm “adipose-derived stem cells” (ASCs) to identify the isolated,plastic-adherent, multipotent cell population. Thus, the term“Adipose-derived stem cell” (ASC) is used herein in accordance with theIFATS consensus, which will be known to one of ordinary skill in theart. In contrast to cell lines, ASCs have not undergone immortalization.

A number of scientific publications have described the underlyingbiology of ASCs, preclinical studies for the use of ASCs in regenerativemedicine in various fields have been performed, and the efficacy of ASCshas been determined in several clinical trials.

ASCs for use in the subject methods can be isolated by any convenientmethods, which will be known to one of ordinary skill in the art.Subcutaneous adipose tissue samples can generally be obtained underlocal anesthesia. Current methods used for isolating ASCs generallyinclude collagenase digestion followed by centrifugal separation toisolate the Stromal Vascular Fraction from primary adipocytes.

As a non-limiting example, to isolate ASCs, adipose tissue obtained asexcised surgical specimens (e.g., a biopsy) or as lipoaspirates can bedigested with a collagenase enzyme (e.g., a bacterially-derivedcollagenase) in the presence of calcium to release the individual cellcomponents. Subsequently, mature adipocytes can be separated (e.g., viadifferential centrifugation), from the remaining cells, which form aStromal Vascular Fraction (SVF) pellet. The SVF cell population includesendothelial cells, fibroblasts, Band T-lymphocytes, macrophages, myeloidcells, pericytes, pre-adipocytes, smooth muscle cells, and the cultureadherent ASCs. After culture (e.g., 4 to 6 days) with medium containingabout 10% fetal bovine serum (any convenient culture medium can beused), a single milliliter of human lipoaspirate will yield between 0.25to 0.375×10⁶ ASCs capable of differentiating along the adipogenic(adipocyte), chondrogenic (chondrocyte), and osteogenic (osteoblast)lineages in vitro. ASCs display a fibroblast-like morphology and lackthe intercellular lipid droplets seen in adipocytes. Isolated ASCs aretypically expanded in monolayer culture on standard tissue cultureplastics with a basal medium containing 10% fetal bovine serum. Sinceliposuction from a single patient often results in >1 L of tissue, it isfeasible to generate hundreds of millions of ASCs from a single donorwithin a single in vitro cell culture passage In contrast to the SVFcells, ASCs are relatively homogeneous based on their expression profileof surface antigens.

In general, the isolation of ASCs from a lipoaspirate can include: (1)wash the lipoaspriate in buffered saline solution; (2) subject thelipoaspirate to collagenase digestion; (3) centrifuge and isolate thestromal vascular fraction (SVF) pellet; (4) culture the heterogeneousSVF cells on an adherent surface; and (5) isolate adherent ASCs. Cultureof SVF cells under standard conditions eventually (within the first fewpassages) results in the appearance of ASCs, which is a relativelyhomogeneous population of mesodermal or mesenchymal cells. ASCs can beverified, for example, by demonstrating that the cells can differentiateinto multiple different lineages (e.g., adipocytes can be identifiedusing, for example, Oil Red O stain; osteoblasts can be identifiedusing, for example, Alizarin Red stain; and chondrocytes can beidentified using, for example, Alcian Blue stain). Protein and nucleicacid markers can also be used to verify differentiation into multiplelineages.

The International Society for Cellular Therapy (ISCT) and IFATS haveestablished minimal criteria defining SVF cells and ASC based onfunctional and quantitative criteria. The four criteria used herein are:(1) ASCs are plastic-adherent when maintained under standard cultureconditions; (2) ASCs have the capacity for osteogenic, adipogenic, andchondrogenic differentiation; (3) ASCs express the markers (i.e.,molecular markers) CD29, CD34, CD36, CD49f, CD73, CD90 (Thy-1), CD105,CD133, c-kit, and c-met; and (4) ASCs are negative for CD45, CD106, andCD31. The adipocytic, chondroblastic and osteoblastic differentiationassays (e.g., Oil Red O stain, Alcian blue stain, and Alizarin redstain, respectively) can be used to assess potency and differentiationcapacity, and can be used in conjunction with a quantitative evaluationof differentiation either biochemically or by reverse transcriptionpolymerase chain reaction. The colony-forming unit-fibroblast (CFU-F)assay is recommended by the IFATS to calculate population doublingscapacity of ASCs.

For more information regarding the nature of ASCs, including theisolation and culture of ASCs, see (i) Gimble et al., Circ Res. 2007 May11; 100(9):1249-60: “Adipose-derived stem cells for regenerativemedicine”; (ii) Gimble et al., Organogenesis. 2013 Jan. 1; 9(1):“Adipose-derived stromal/stem cells: A primer”; (iii) Bourin et al,Cytotherapy. 2013 Jun.; 15(6):641-8: “Stromal cells from the adiposetissue-derived stromal vascular fraction and culture expanded adiposetissue-derived stromal/stem cells: a joint statement of theInternational Federation for Adipose Therapeutics and Science (IFATS)and the International Society for Cellular Therapy (ISCT)”; (iv) Gentileet al, Stem Cells Transl Med. 2012 Mar.; 1(3):230-6: “Concise review:adipose-derived stromal vascular fraction cells and platelet-richplasma: basic and clinical implications for tissue engineering therapiesin regenerative surgery”; and (v) Mizuno et al., Stem Cells. 2012 May;30(5):804-10: “Concise review: Adipose-derived stem cells as a noveltool for future regenerative medicine”; all of which are herebyincorporated by reference in their entirety.

As used herein, the term “adipose tissue” refers to fat including theconnective tissue that stores fat. Adipose tissue contains multipleregenerative cell types, including ASCs and endothelial progenitor andprecursor cells.

Three dimensional culture. Methods of the disclosure include placing apopulation of ASCs into a three dimensional culture to produce anASC-derived cellular aggregate (e.g., sphere). The term “threedimensional culture” as used herein refers to the culture of cells in away that includes low shear force (and consequently low turbulence), andhigh mass transfer of nutrients. One non-limiting example of “threedimensional culture” is the culture of cells as aggregates (e.g.,spheres, i.e., spheroid formation). Placing a population of cells (e.g.,ASCs) into a three dimensional culture can cause the cells to formcellular aggregates (e.g., cellular spheres). Thus, subject cells (e.g.,a population of ASCs) can be placed into a three dimensional culture toproduce an ASC-derived cellular aggregate (e.g, sphere). Cellularaggregates (e.g, spheres) can be produced by a number of threedimensional culture techniques, including but not limited to hangingdrop suspension culture, high density cell culture (e.g., stirredsuspension culture such as spinner flask culture, e.g., with or withoutmicrocarriers; bioreactor culture, e.g., with or without microcarriers;etc.), and the like (see examples section).

In some embodiments, cells can be placed into a three dimensionalculture at any convenient chosen density (e.g., via dilution orconcentration). For example, in some cases, cells are placed into athree dimensional culture at a cell density in a range of from 1×10²cells/ml to 1×10⁷ cells/ml (e.g., from 5×10² cells/ml to 5×10⁶ cells/ml,from 1×10³ cells/ml to 5×10⁶ cells/ml, from 5×10³ cells/ml to 5×10⁶cells/ml, from 5×10³ cells/ml to 1×10⁵ cells/ml, from 5×10³ cells/ml to5×10⁴ cells/ml, from 7×10³ cells/ml to 3×10⁴ cells/ml, from 8×10³cells/ml to 3×10⁴ cells/ml, 1×10⁴ cells/ml, from 1×10⁴ cells/ml to 1×10⁶cells/ml, from 5×10⁴ cells/ml to 1×10⁶ cells/ml, from 7×10⁴ cells/ml to7×10⁵, from 1×10⁵ cells/ml to 7×10⁵, from 3×10⁵ cells/ml to 7×10⁵, from4×10⁵ cells/ml to 6×10⁵, or 5×10⁵).

In practicing the methods of the disclosure, ASCs can be cultured inthree dimensional culture for a period of time sufficient for theformation of cellular aggregates (e.g., spheres, which can resembleembryoid bodies). In some embodiments, ASCs are cultured using threedimensional culture (i.e., ASCs are placed into a three dimensionalculture) for 3 days or less (e.g., 2.5 days or less, 2 days or less, 1.5days or less, 1 day or less, 12 hours or less, 8 hours or less, 6 hoursor less, or 4 hours or less). In some embodiments, ASCs are culturedusing three dimensional culture for a period of time in a range of from2 hours to 3 days (e.g., from 2 hours to 3 days, from 2 hours to 2.5days, from 2 hours to 2 days, from 2 hours to 1.5 days, from 2 hours to1 day, from 6 hours to 3 days, from 6 hours to 2.5 days, from 6 hours to2 days, from 6 hours to 1.5 days, from 6 hours to 1 day, from 6 hours to12 hours, from 8 hours to 3 days, from 8 hours to 2.5 days, from 8 hoursto 2 days, from 8 hours to 1.5 days, from 8 hours to 1 day, from 8 hoursto 12 hours, from 12 hours to 2.5 days, from 12 hours to 2 days, from 12hours to 1.5 days, from 12 hours to 1 day, from 1 day to 3 days, from 1day to 2.5 days, from 1 day to 2 days, from 1 day to 1.5 days, from 1.5days to 3 days, from 1.5 days to 2.5 days, from 1.5 days to 2 days, from2 days to 3 days, from 1 day, 1.5 days, from 2 days, from 2.5 days, or 3days).

Hanging drop suspension culture. In some embodiments, the threedimensional culture is a hanging drop suspension culture. Thus, in someembodiments, aggregates of subject cells (e.g., ASCs) are formed byplacing the cells in a hanging drop suspension culture. Hanging dropsuspension culture is a technique often used to form embryoid bodiesfrom embryonic stem cells (ESCs). Cells in fluid are placed in droplets(usually in the range of 5-50 μl in size) onto a surface (e.g., surfaceof a Petri dish, a coverslip, glass, plastic etc.) and the surface isinverted such that the cells are suspended from the surface. The cellsare thus cultured under the surface in a suspended droplet instead ofbeing cultured on top of the surface. The suspended droplets aresometimes referred to as hanging drops. Some cells form aggregates,(referred to as ‘spheres’ and/or ‘spheroids’) when cultured using thehanging drop suspension culture.

In some embodiments, cells can be placed into hanging drop suspensionculture at a chosen density (e.g., via dilution or concentration). Forexample, in some cases, cells are placed into hanging drop suspensionculture at a cell density in a range of from 1×10² cells/ml to 1×10⁷cells/ml (e.g., from 5×10² cells/ml to 5×10⁶ cells/ml, from 1×10³cells/ml to 5×10⁶ cells/ml, from 5×10³ cells/ml to 5×10⁶ cells/ml, from5×10³ cells/ml to 1×10⁵ cells/ml, from 5×10³ cells/ml to 5×10⁴ cells/ml,from 7×10³ cells/ml to 3×10⁴ cells/ml, from 8×10³ cells/ml to 3×10⁴cells/ml, 1×10⁴ cells/ml, from 1×10⁴ cells/ml to 1×10⁶ cells/ml, from5×10⁴ cells/ml to 1×10⁶ cells/ml, from 7×10⁴ cells/ml to 7×10⁵, from1×10⁵ cells/ml to 7×10⁵, from 3×10⁵ cells/ml to 7×10⁵, from 4×10⁵cells/ml to 6×10⁵, or 5×10⁵).

In practicing the methods of the disclosure, ASCs can be cultured by thehanging drop method (i.e., cultured using hanging drop suspensionculture) for a period of time sufficient for the formation of cellularaggregates (e.g., spheres, which can resemble embryoid bodies). In someembodiments, ASCs are cultured using hanging drop suspension culture(i.e., ASCs are placed into a hanging drop suspension culture) for 3days or less (e.g., 2.5 days or less, 2 days or less, 1.5 days or less,1 day or less, 12 hours or less, 8 hours or less, 6 hours or less, or 4hours or less). In some embodiments, ASCs are cultured using hangingdrop suspension culture for a period of time in a range of from 2 hoursto 3 days (e.g., from 2 hours to 3 days, from 2 hours to 2.5 days, from2 hours to 2 days, from 2 hours to 1.5 days, from 2 hours to 1 day, from6 hours to 3 days, from 6 hours to 2.5 days, from 6 hours to 2 days,from 6 hours to 1.5 days, from 6 hours to 1 day, from 6 hours to 12hours, from 8 hours to 3 days, from 8 hours to 2.5 days, from 8 hours to2 days, from 8 hours to 1.5 days, from 8 hours to 1 day, from 8 hours to12 hours, from 12 hours to 2.5 days, from 12 hours to 2 days, from 12hours to 1.5 days, from 12 hours to 1 day, from 1 day to 3 days, from 1day to 2.5 days, from 1 day to 2 days, from 1 day to 1.5 days, from 1.5days to 3 days, from 1.5 days to 2.5 days, from 1.5 days to 2 days, from2 days to 3 days, from 1 day, 1.5 days, from 2 days, from 2.5 days, or 3days).

In some embodiments, an ASC-derived cellular aggregate (e.g., sphere) isremoved from three dimensional culture (e.g., hanging drop suspensionculture, high density culture, spinner flask culture, microcarrierculture, etc.). For example, a cellular aggregate (e.g., sphere) can besuspended in fluid and plated onto a two-dimensional surfaced that isnot considered a three dimensional culture. In some cases, aggregates(e.g., spheres) are collected (e.g., by centrifugation) and suspended.In some cases, the aggregates (e.g., spheres) are collected andsuspended at a density in a range of from 15 aggregates/ml to 45aggregates/ml (e.g., from 20 aggregates/ml to 40 aggregates/ml, from 25aggregates/ml to 35 aggregates/ml, or 30 aggregates/ml). The aggregatescan be suspended in any convenient media (e.g., stage 1 media, describedbelow). In some cases, the collected (suspended) aggregates (e.g.,spheres) are seeded (i.e., plated, e.g., onto matrigel-coated dishes).In some embodiments, an ASC-derived cellular aggregate (e.g., sphere) isremoved from three dimensional culture (e.g., hanging drop suspensionculture, high density culture, spinner flask culture, microcarrierculture, etc.) simultaneous with, or prior to, contacting cells of theASC-derived cellular aggregate (e.g., sphere) with a stage 1 medium.Thus, in some cases, cells are cultured (e.g., contacted with) a stage 1medium after being removed from three dimensional culture. In someembodiments, cells of the ASC-derived cellular aggregate (e.g., sphere)are contacted with a stage 1 medium while still in three dimensionalculture (e.g., in order to transfer the cells to non-inverted cultureconditions).

High density culture—microcarrier culture. In some embodiments, thethree dimensional culture is a high density culture. Thus, aggregates(e.g., spheres) of subject cells (e.g., ASCs) are formed by placing thecells in a high density culture. In some embodiments, a high densityculture is a microcarrier culture. In some embodiments, the threedimensional culture is a microcarrier culture. Thus, aggregates (e.g.,spheres) of subject cells (e.g., ASCs) are formed by placing the cellsin a microcarrier culture. The term “microcarrier culture” is usedherein to refer to the culture of cells on a support matrix (e.g., aspherical support matrix). In this system, cells are propagated on thesurface of small solid particles suspended in the growth medium by slowagitation. The cells attach and grow to confluence on the surface of themicrocarriers.

Microcarriers can be produced in various shapes and sizes, sphericalbeing the most common, and their density allows them to be maintained insuspension with gentle stirring. Each microcarrier should havedimensions that can facilitate cell growth for several doublings. Inthis way at the end of the cell growth, each microcarrier will supportseveral hundred cells on its surface. Spherical microcarriers usuallyhave diameters of 100-250 μm. In some embodiments, microcarriers arespheres with a diameter in a range of from 100 μm to 250 μm. The sizedistribution of the spherical microcarriers should be low (e.g., ±25 μm)in order to prevent uneven distribution of cells on the microcarriers.Density of the microcarriers should be slightly above 1 (e.g., 1.02-1.05g/ml) in order to maintain the microcarriers in suspension at minimalagitation speed.

Microcarriers can be made from a number of different materials includingdiethylaminoethanol (DEAE)-dextran, dextran, glass, polystyrene plastic,acrylamide (e.g., polyacrylamide), collagen, etc. In some cases,biodegradable microcarriers can be used as the scaffold for in vivotransplantation of cells. The MC surface is available for cell growthwhile the mobility of MCs in the medium generates a homogeneity that issimilar to the suspension environment used in traditional mammalian andmicrobial submerged cultures.

The surface of a microcarrier can be derivatized with functional groupssuch as recombinant proteins, positively charged tertiary quaternary orprimary amines, gelatin, collagen, other extracellular matrix (ECM)proteins and peptides (e.g. RGD peptide). The positively charged MCsattract the cells (which are negatively charged), by electrostaticforces. The optimal amount of positive charge is generally found to bebetween 1 and 2 milliequivalents/g dry materials (for cross-linkeddextran or polyacrylamide beads derivatized with tertiary amines). Atthis level, cells attach to the microcarriers efficiently (about 90%within 1 h) without negative effect on cell growth. Coating withcollagen or ECM protein results in lower cell attachment but usuallysupports better growth of cells at low inoculation levels.

Several types of microcarriers are available commercially includingdextran-based (Cytodex, GE Healthcare), collagen-based (Cultispher,Percell), and polystyrene-based (SoloHill Engineering) microcarriers.They differ in their porosity, specific gravity, optical properties,presence of animal components, and surface chemistries. It is possibleto categorize the microcarriers into six groups:

Group 1: Non-porous smooth (e.g. polystyrene microcarriers) ormicroporous microcarriers (e.g. Cytodex 1) with positive charges. Thesemicrocarriers are suitable for culturing adherent cells that form acontinuous monolayer of cells on the surface of the microcarriers instirred cultures. This group includes also Whatman's anion exchangecelluloses (DE-53), cylindrical shaped microcarriers that have been usedsuccessfully in culturing cell lines (BHK and MDCK).

Group 2: Collagen coated microcarriers (e.g. Cytodex 3 and FACT 102-L).These microcarriers are chemically coupled with collagen and aresuitable for culturing sensitive cells with low plating efficiency. Thecollagen coating is also designed to facilitate cell harvesting.

Group 3: ECM coated microcarriers (Pro-F 102-L). Pro-F 102-L is coatedwith recombinant fibronectin which is designed for culturing ofsensitive cells in serum free conditions.

Group 4: Non-charged microcarriers (e.g. Glass beads and tissue culturePolystyrene MC P 102-L). These microcarriers have similar surfaceproperties as classical 2D tissue culture surfaces.

Group 5: Macroporous microcarriers (e.g. Cytopore and Cultispher).Macroporous microcarriers with pore sizes in the range of 10-70 μm onthe surface. They provide higher cell surface areas for growth and offerbetter mechanical protection to the cells from shear stress generated bystirrers, spargers or spin filters.

Group 6: Weighted microcarriers (Cytoline). These microcarriers aredesigned for use in fluidized bed perfusion cultures. These commercialmicrocarriers have been designed according to the needs for propagatinganchorage dependent cell lines used in production of vaccine andbiopharmaceuticals.

In practicing the methods of the disclosure, ASCs can be cultured bymicrocarrier culture (i.e., cultured using microcarrier culture) foraperiod of time sufficient for the formation of cellular aggregates(e.g., spheres, which can resemble embryoid bodies). In someembodiments, ASCs are cultured using microcarrier culture (i.e., ASCsare placed into a microcarrier culture) for 3 days or less (e.g., 2.5days or less, 2 days or less, 1.5 days or less, 1 day or less, 12 hoursor less, 8 hours or less, 6 hours or less, or 4 hours or less). In someembodiments, ASCs are cultured using microcarrier culture for a periodof time in a range of from 2 hours to 3 days (e.g., from 2 hours to 3days, from 2 hours to 2.5 days, from 2 hours to 2 days, from 2 hours to1.5 days, from 2 hours to 1 day, from 6 hours to 3 days, from 6 hours to2.5 days, from 6 hours to 2 days, from 6 hours to 1.5 days, from 6 hoursto 1 day, from 6 hours to 12 hours, from 8 hours to 3 days, from 8 hoursto 2.5 days, from 8 hours to 2 days, from 8 hours to 1.5 days, from 8hours to 1 day, from 8 hours to 12 hours, from 12 hours to 2.5 days,from 12 hours to 2 days, from 12 hours to 1.5 days, from 12 hours to 1day, from 1 day to 3 days, from 1 day to 2.5 days, from 1 day to 2 days,from 1 day to 1.5 days, from 1.5 days to 3 days, from 1.5 days to 2.5days, from 1.5 days to 2 days, from 2 days to 3 days, from 1 day, 1.5days, from 2 days, from 2.5 days, or 3 days).

For examples of the use of microcarriers (e.g., using spinner flasks,bioreactors, etc.) for various purposes and with various types of cells,refer to scientific literature such as: (i) Chen et al, Biotechnol Adv.2013 Mar. 24. pii: S0734-9750(13)00065-7: Application of humanmesenchymal and pluripotent stem cell microcarrier cultures in cellulartherapy: Achievements and future direction; (ii) Pacak et al, PLoS One.2013; 8(1):e55187: Microcarrier-based expansion of adult murine sidepopulation stem cells; (iii) Torgan et al, Med Biol Eng Comput. 2000September; 38(5):583-90: Differentiation of mammalian skeletal musclecells cultured on microcarrier beads in a rotating cell culture system;and (iv) Park et al, Tissue Eng Part B Rev. 2013 Apr.; 19(2):172-90:Microcarriers designed for cell culture and tissue engineering of bone;as well as patent literature such as U.S. Pat. Nos. 8,524,492,8,426,176, 7,947,471, 7,670,839, 7,361,493, 6,214,618, and 5,153,133,4,910,142, and 4,824,946; all of which are hereby incorporated byreference in their entirety.

Microcarrier culture generally requires agitation of the microcarriers(e.g., the cell-loaded microcarriers). Agitation can include simpleagitation or shaking, agitation using a spinner flask, and/or agitationusing a bioreactor (also known as a rotating cell culture system (RCCS),a rotary cell culture system (RCCS), or a rotating chamber system).

High density culture—Spinner flask culture. In some embodiments, thethree dimensional culture is a high density culture. Thus, aggregates(e.g., spheres) of subject cells (e.g., ASCs) are formed by placing thecells in a high density culture. In some embodiments, a high densityculture is a spinner flask culture. Spinner flask culture is an exampleof a stirred suspension culture. Stirred suspension culture (i.e., masssuspension culture)(e.g., spinner flask culture, bioreactor culture,etc.) can be used to culture cells in the absence of microcarriers or inthe presence of microcarriers. Thus, in some embodiments, a microcarrierculture is agitated using a spinner flask. In some embodiments, a threedimensional culture (e.g., a high density culture) is a spinner flaskculture without (i.e., in the absence of) microcarriers. In the absenceof microcarriers, ASCs placed in stirred suspension culture (e.g.,spinner flask culture, bioreactor culture) still form cellularaggregates (e.g., spheres)(FIG. 13). Thus, in some embodiments, a threedimensional culture (e.g., a high density culture) is a spinner flaskculture without (i.e., in the absence of) microcarriers.

Spinner flasks (i.e., stirrer bottles) are generally designed tofacilitate the culture of high volumes of cells by providing homogenouscirculation of cells (e.g., cells in the absence of microcarriers, cellson microcarriers, etc.) and medium (i.e., nutrients), and superior gasexchange (e.g., oxygenation). Spinner flasks can be made of anyconvenient material (e.g., borosilicate glass, tissue culture gradeplastics, etc.) and are usually a flat-bottom flask with a stirringdevice (e.g., usually a vertical impeller or a hanging stir bar) thatcan be controlled using a magnetic stir plate. A spinner flask can alsoinclude two angled side arms to allow for the introduction of pipettes.Any convenient spinner flask that provides for high density culture canbe used in practicing the subject methods.

In some embodiments, cells can be placed into a spinner flask culture atany convenient chosen density (e.g., via dilution or concentration). Forexample, in some cases, cells are placed into a spinner flask culture ata cell density in a range of from 1×10² cells/ml to 1×10⁷ cells/ml(e.g., from 5×10² cells/ml to 5×10⁶ cells/ml, from 1×10³ cells/ml to5×10⁶ cells/ml, from 5×10³ cells/ml to 5×10⁶ cells/ml, from 5×10³cells/ml to 1×10⁵ cells/ml, from 5×10³ cells/ml to 5×10⁴ cells/ml, from7×10³ cells/ml to 3×10⁴ cells/ml, from 8×10³ cells/ml to 3×10⁴ cells/ml,1×10⁴ cells/ml, from 1×10⁴ cells/ml to 1×10⁶ cells/ml, from 5×10⁴cells/ml to 1×10⁶ cells/ml, from 7×10⁴ cells/ml to 7×10⁵, from 1×10⁵cells/ml to 7×10⁵, from 3×10⁵ cells/ml to 7×10⁵, from 4×10⁵ cells/ml to6×10⁵, or 5×10⁵).

In practicing the methods of the disclosure, ASCs can be cultured byspinner flask culture (i.e., cultured using spinner flask culture) foraperiod of time sufficient for the formation of cellular aggregates(e.g., spheres, which can resemble embryoid bodies). In someembodiments, ASCs are cultured using spinner flask culture (i.e., ASCsare placed into a spinner flask culture) for 3 days or less (e.g., 2.5days or less, 2 days or less, 1.5 days or less, 1 day or less, 12 hoursor less, 8 hours or less, 6 hours or less, or 4 hours or less). In someembodiments, ASCs are cultured using spinner flask culture for a periodof time in a range of from 2 hours to 3 days (e.g., from 2 hours to 3days, from 2 hours to 2.5 days, from 2 hours to 2 days, from 2 hours to1.5 days, from 2 hours to 1 day, from 6 hours to 3 days, from 6 hours to2.5 days, from 6 hours to 2 days, from 6 hours to 1.5 days, from 6 hoursto 1 day, from 6 hours to 12 hours, from 8 hours to 3 days, from 8 hoursto 2.5 days, from 8 hours to 2 days, from 8 hours to 1.5 days, from 8hours to 1 day, from 8 hours to 12 hours, from 12 hours to 2.5 days,from 12 hours to 2 days, from 12 hours to 1.5 days, from 12 hours to 1day, from 1 day to 3 days, from 1 day to 2.5 days, from 1 day to 2 days,from 1 day to 1.5 days, from 1.5 days to 3 days, from 1.5 days to 2.5days, from 1.5 days to 2 days, from 2 days to 3 days, from 1 day, 1.5days, from 2 days, from 2.5 days, or 3 days).

High density culture—Bioreactor culture. In some embodiments, the threedimensional culture is a high density culture. Thus, aggregates (e.g.,spheres) of subject cells (e.g., ASCs) are formed by placing the cellsin a high density culture. In some embodiments, a high density cultureis a bioreactor culture. Bioreactor culture is an example of a stirredsuspension culture. Stirred suspension culture (i.e., mass suspensionculture)(e.g., spinner flask culture, bioreactor culture, etc.) can beused to culture cells in the absence of microcarriers or in the presenceof microcarriers. Thus, in some embodiments, a microcarrier culture isagitated using a bioreactor. In some embodiments, a three dimensionalculture (e.g., a high density culture) is a bioreactor culture without(i.e., in the absence of) microcarriers. In the absence ofmicrocarriers, ASCs placed in stirred suspension culture (e.g., spinnerflask culture, bioreactor culture) still form cellular aggregates (e.g.,spheres). Thus, in some embodiments, a three dimensional culture (e.g.,a high density culture) is a bioreactor culture without (i.e., in theabsence of) microcarriers.

Bioreactors (also known as a rotating cell culture system (RCCS), arotary cell culture system (RCCS), or a rotating chamber system) cancontrol environmental conditions such as gas (e.g., air, oxygen,nitrogen, carbon dioxide) flow rates, temperature, pH, dissolved oxygenlevels, agitation speed/circulation rate, and the like. Like spinnerflasks, bioreactors are designed to facilitate the culture of highvolumes of cells by providing homogenous circulation of cells (e.g.,cells in the absence of microcarriers, cells on microcarriers, etc.) andmedium (i.e., nutrients), and superior gas exchange (e.g., oxygenation).Any convenient bioreactor that provides for high density cell culturecan be used with the disclosed methods and various suitable bioreactorsare known in the art. Examples of suitable biorecactors include, but arenot limited to: rotating wall microgravity bioreactors, rotating wallvessel bioreactors (RWVB), stirred tank bioreactors, perfusedbioreactors, fluidized bed bioreactors. Bioreactors can be made from alarge variety of different materials, including, but not limited to:stainless steel, polytetraflouroethylene (PTFE), glass, and the like.

Bioreactors can allow for the scaling up of culture in, for example,conventional stainless steel or disposable bioreactors that are used forpropagation of suspended mammalian cells. Bioreactors range in size. Insome cases a bioreactor culture is a culture in the range of from 5liters to 105 liters (e.g., from 5 liters to 15 liters, from 8 liters to12 liters, from 15 liters to 25 liters, from 25 liters to 40 liters,from 40 liters to 50 liters, from 45 liters to 55 liters, from 55 litersto 65 liters, from 65 liters to 75 liters, from 75 liters to 85 liters,from 85 liters to 95 liters, or from 95 liters to 105 liters).

In some embodiments, cells can be placed into a bioreactor culture atany convenient chosen density (e.g., via dilution or concentration). Forexample, in some cases, cells are placed into a bioreactor culture at acell density in a range of from 1×10² cells/ml to 1×10⁷ cells/ml (e.g.,from 5×10² cells/ml to 5×10⁶ cells/ml, from 1×10³ cells/ml to 5×10⁶cells/ml, from 5×10³ cells/ml to 5×10⁶ cells/ml, from 5×10³ cells/ml to1×10⁵ cells/ml, from 5×10³ cells/ml to 5×10⁴ cells/ml, from 7×10³cells/ml to 3×10⁴ cells/ml, from 8×10³ cells/ml to 3×10⁴ cells/ml, 1×10⁴cells/ml, from 1×10⁴ cells/ml to 1×10⁶ cells/ml, from 5×10⁴ cells/ml to1×10⁶ cells/ml, from 7×10⁴ cells/ml to 7×10⁵, from 1×10⁵ cells/ml to7×10⁵, from 3×10⁵ cells/ml to 7×10⁵, from 4×10⁵ cells/ml to 6×10⁵, or5×10⁵).

In practicing the methods of the disclosure, ASCs can be cultured inbioreactor culture for a period of time sufficient for the formation ofcellular aggregates (e.g., spheres, which can resemble embryoid bodies).In some embodiments, ASCs are cultured using bioreactor culture (i.e.,ASCs are placed into a bioreactor culture) for 3 days or less (e.g., 2.5days or less, 2 days or less, 1.5 days or less, 1 day or less, 12 hoursor less, 8 hours or less, 6 hours or less, or 4 hours or less). In someembodiments, ASCs are cultured using bioreactor culture fora period oftime in a range of from 2 hours to 3 days (e.g., from 2 hours to 3 days,from 2 hours to 2.5 days, from 2 hours to 2 days, from 2 hours to 1.5days, from 2 hours to 1 day, from 6 hours to 3 days, from 6 hours to 2.5days, from 6 hours to 2 days, from 6 hours to 1.5 days, from 6 hours to1 day, from 6 hours to 12 hours, from 8 hours to 3 days, from 8 hours to2.5 days, from 8 hours to 2 days, from 8 hours to 1.5 days, from 8 hoursto 1 day, from 8 hours to 12 hours, from 12 hours to 2.5 days, from 12hours to 2 days, from 12 hours to 1.5 days, from 12 hours to 1 day, from1 day to 3 days, from 1 day to 2.5 days, from 1 day to 2 days, from 1day to 1.5 days, from 1.5 days to 3 days, from 1.5 days to 2.5 days,from 1.5 days to 2 days, from 2 days to 3 days, from 1 day, 1.5 days,from 2 days, from 2.5 days, or 3 days).

Culturing cells in differentiation media. The term “differentiationmedia” as used herein refers to media can be used to induce cells todifferentiate. For example, stage 1 and stage 2 media (described below)are two types of differentiation media used to induce ASCs todifferentiate into hepatocyte-like cells. In some embodiments, themethods include removing the ASC-derived cellular aggregate (e.g.,sphere) from three dimensional culture (e.g., hanging drop suspensionculture, high density culture, spinner flask culture, bioreactorculture, stirred suspension culture, microcarrier culture, etc.) andcontacting cells of the ASC-derived cellular aggregate (e.g., for 3 daysor less) with a first culture medium (e.g., stage 1 media) comprisingActivin A and a fibroblast growth factor (FGF) to produce a precursorcell population. In some embodiments, cells of the precursor cellpopulation are contacted with a second culture medium (e.g., stage 2media) comprising hepatocyte growth factor (HGF) (e.g., for 7 days orless) to produce an induced cell population that comprises inducedhepatocyte-like cells (iHeps). All proteins listed below that can beincluded in a suitable differentiation media, can be provided from anyconvenient source (e.g., purified from tissue, recombinant, etc.).

In some embodiments, cells of an ASC-derived cellular aggregate (e.g.,sphere) are contacted with stage 1 media (as defined below, e.g., aculture medium having Activin A and an FGF) for 3 days or less (e.g.,2.5 days or less, 2 days or less, 1.5 days or less, 1 day or less, or 12hours or less) to produce a precursor cell population. In someembodiments, cells of an ASC-derived cellular aggregate (e.g., sphere)are contacted with stage 1 media for a period of time in a range of from12 hours to 3 days (e.g., 12 hours to 2.5 days, 12 hours to 2 days, 12hours to 1.5 days, 12 hours to 1 day, 1 day to 3 days, 1 day to 2.5days, 1 day to 2 days, 1 day to 1.5 days, 1.5 days to 3 days, 1.5 daysto 2.5 days, 1.5 days to 2 days, 2 days to 3 days, 1 day, 1.5 days, 2days, 2.5 days, or 3 days) to produce a precursor cell population.

In some embodiments, cells of the precursor cell population arecontacted with stage 2 media (as defined below, e.g., a culture mediumhaving hepatocyte growth factor (HGF)) for 8 days or less (e.g., 7.5days or less, 7 days or less, 6.5 days or less, 6 days or less, 5.5 daysor less, 5 days or less, 4.5 days or less, 4 days or less, 3.5 days orless, 3 days or less, 2.5 days or less, 2 days or less, 1.5 days orless, 1 day or less, or 12 hours or less) to produce an induced cellpopulation that comprises induced hepatocyte-like cells. In someembodiments, cells of the precursor cell population are contacted withstage 2 media fora period of time in a range of from 12 hours to 8 days(e.g., 12 hours to 8 days, 1 day to 8 days, 2 days to 8 days, 3 days to8 days, 4 days to 8 days, 5 days to 8 days, 6 days to 8 days, 2 days to7 days, 3 days to 7 days, 4 days to 7 days, 5 days to 7 days, 6 days to7 days, 5.5 days to 8 days, 5.5 days to 7.5 days, 5.5 days to 6.5 days,6 days to 7.5 days, 6.5 days to 7.5 days, 1 day, 2 days, 3 days, or 3days) to produce an induced cell population that comprises inducedhepatocyte-like cells.

In some embodiments, the total time elapsed from (i) placing apopulation of ASCs into a three dimensional culture, to (ii) producingan induced cell population that comprises induced hepatocyte-like cells(iHeps), is less than 13 days (e.g., less than 12.5 days, less than 12days, less than 11.5 days, less than 11 days, less than 10.5 days, lessthan 10 days, less than 9.5 days, less than 9 days, less than 8.5 days,less than 8 days, less than 7.5 days, less than 7 days, less than 6.5days, less than 6 days, less than 5.5 days, less than 5 days, less than4.5 days, or less than 4 days).

In some embodiments, the total time elapsed from (i) placing apopulation of ASCs into a three dimensional culture, to (ii) producingan induced cell population that comprises induced hepatocyte-like cells(iHeps), is 13 days or less (e.g., 12.5 days or less, 12 days or less,11.5 days or less, 11 days or less, 10.5 days or less, 10 days or less,9.5 days or less, 9 days or less, 8.5 days or less, 8 days or less, 7.5days or less, 7 days or less, 6.5 days or less, 6 days or less, 5.5 daysor less, 5 days or less, 4.5 days or less, or 4 days or less).

In some embodiments, the total time elapsed from (i) placing apopulation of ASCs into a three dimensional culture, to (ii) producingan induced cell population that comprises induced hepatocyte-like cells(iHeps), is in a range of from 12 hours to 13 days (e.g., from 1 day to13 days, from 2 days to 13 days, from 3 days to 13 days, from 4 days to13 days, from 4.5 days to 13 days, from 5 days to 13 days, from 5.5 daysto 13 days, from 6 days to 13 days, from 6.5 days to 13 days, from 7days to 13 days, from 7.5 days to 13 days, from 8 days to 13 days, from8.5 days to 13 days, from 9 days to 13 days, from 9.5 days to 13 days,from 10 days to 13 days, from 10.5 days to 13 days, from 11 days to 13days, from 11.5 days to 13 days, from 12 hours to 12.5 days, from 1 dayto 12.5 days, from 2 days to 12.5 days, from 3 days to 12.5 days, from 4days to 12.5 days, from 4.5 days to 12.5 days, from 5 days to 12.5 days,from 5.5 days to 12.5 days, from 6 days to 12.5 days, from 6.5 days to12.5 days, from 7 days to 12.5 days, from 7.5 days to 12.5 days, from 8days to 12.5 days, from 8.5 days to 12.5 days, from 9 days to 12.5 days,from 9.5 days to 12.5 days, from 10 days to 12.5 days, from 10.5 days to12.5 days, from 11 days to 12.5 days, from 11.5 days to 12.5 days, from12 hours to 12 days, from 1 day to 12 days, from 2 days to 12 days, from3 days to 12 days, from 4 days to 12 days, from 4.5 days to 12 days,from 5 days to 12 days, from 5.5 days to 12 days, from 6 days to 12days, from 6.5 days to 12 days, from 7 days to 12 days, from 7.5 days to12 days, from 8 days to 12 days, from 8.5 days to 12 days, from 9 daysto 12 days, from 9.5 days to 12 days, from 10 days to 12 days, from 10.5days to 12 days, from 11 days to 12 days, from 3 days to 11 days, from 3days to 10 days, from 3 days to 9.5 days, from 3 days to 9 days, from 6days 10 days, or from 4 days to 10 days).

Basal cell culture media. Subject cells (e.g., ASCs, iHeps,differentiating ASCs, etc.) are cultured in an appropriate liquidnutrient medium, referred to herein as “basal cell culture media.”. Thestage 1 and stage 2 media are basal culture media supplemented with atleast one additional component, as described below. Various basal mediaformulations are available (e.g., Dulbecco's Modified Eagle Medium(DMEM), RPMI, Iscove's medium, hepatocyte culture medium (HCM) (Lonza,Cat: cc-3198) etc.).

Any convenient culture media can serve as a basal culture medium. Forexample, a suitable tissue culture medium can contain components such asa vitamin; an amino acid (e.g., an essential amino acid, a non-essentialamino acid, etc.); a pH buffering agent; a salt; an antimicrobial agent(e.g., an antibacterial agent, an antimycotic agent, etc.); serum (e.g.,fetal bovine serum, human serum, calf serum, horse serum, goat serumetc.); an energy source (e.g., a sugar); a nucleoside; a lipid; tracemetals; a cytokine; a growth factor; a stimulatory factor; additives(e.g., pyruvate (0.1-5 mM), glutamine (0.5-5 mM), etc.) and the like.Any convenient cell culture media can be used, and as is known in theart, various cell types grow better in particular media preparations.For example, in some cases, particular media formulations have beenoptimized to culture specific types of cells (e.g., ASCs, hepatocytes(hepatocyte culture medium (HCM) (Lonza, Cat: cc-3198)), neuralprogenitors, embryonic stem cells, etc.). Accordingly, any convenientcell culture media can be used as a basal culture media and may betailored to the culture of ASCs and/or their differentiating progeny.

One exemplary, non-limiting, suitable basal medium is RPMI (Roswell ParkMemorial Institute) 1640, which is well known in the art as a basalmedium that allows the culture of mammalian cells with serum (FetalBovine Serum (FBS)) supplementation. Another suitable basal culturemedia is Advanced RPMI 1640, which is similar to classic RPMI 1640, butallows for serum supplementation to be reduced by 50-90% with no changein growth rate or morphology. Advanced RPMI 1640 can contain, forexample, glucose, non-essential amino acids, sodium pyruvate, and phenolred. The complete formulation is readily available (e.g., online) to oneof ordinary skill in the art for RPMI 1640, for Advanced RPMI 1640, andfor many suitable commercially available basal culture media. Anothersuitable basal culture media is CLONETICS™ hepatocyte culture medium(HCM™) (available, ofr example, from Lonza—Catalog number 3198).

Stage 1 medium. “Stage 1 medium” is also referred to herein as a “firstculture medium.” A suitable stage 1 medium is a basal culture medium(e.g., advanced RPMI 1640, RPMI 1640, etc.) suitable for the culture ofcells (e.g., ASCs, differentiating ASCs, etc.) supplemented with atleast activin A (e.g., 100 ng/ml) and a fibroblast growth factor (e.g.,FGF4, e.g., 20 ng/ml). In some cases, stage 1 medium includes a Wntsignaling agonist (e.g., Wnt3a, e.g., 50 ng/ml). In some cases, stage 1medium includes a supplement such as B27 (e.g., 20%), which is known inthe art. In some cases, a stage 1 medium includes a Wnt signalingagonist and a supplement (e.g., B27). In some cases, the basal culturemedia used for stage 1 media is advanced RPMI 1640 and it issupplemented with activin A (e.g., 100 ng/ml), FGF4 (e.g., 20 ng/ml),Wnt3a (e.g., 50 ng/ml), and 20% B27. In general, Stage 1 medium drivesASC differentiation toward the endoderm lineage.

Activins are dimeric proteins consisting of β subunits, which areconnected by disulfide bonds. There are three different forms ofactivin: (i) homodimeric activin A (2 β_(A) subunits); (ii) homodimericactivin B (2 β_(B) subunits); and (iii) heterodimeric activin AB (1β_(A) and 1 β_(B) subunit). The term “Activin A” as used herein refersto a homodimer of β_(A) subunits. A β_(A) subunit is the polypeptidealso referred to as “Inhibin beta A chain” or “INHBA.” The amino acidsequence of Inhibin beta A chain is:

(SEQ ID NO: 1) MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEEC GCSIn some embodiments, a subject stage 1 medium comprises Activin A at aconcentration in a range of from about 50 ng/ml to about 150 ng/ml(e.g., from about 60 ng/ml to about 140 ng/ml, from about 70 ng/ml toabout 130 ng/ml, from about 80 ng/ml to about 120 ng/ml, from about 85ng/ml to about 115 ng/ml, from about 90 ng/ml to about 110 ng/ml, fromabout 95 ng/ml to about 105 ng/ml, from about 97.5 ng/ml to about 102.5ng/ml, or about 100 ng/ml).

Fibroblast growth factors (FGFs) are a well described family of proteins(related by sequence, structure, and function), with 18 mammalianmembers. The FGFs are grouped into 6 subfamilies based on differences insequence homology and phylogeny: FGFs 1 and 2; FGFs 3, 7, 10, and 22;FGFs 4-6; FGFs 8, 17, and 18; FGFs 9, 16 and 20; and FGFs 19, 21 and 23.In some cases, a suitable FGF is FGF 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 16,17, 18, 20, and/or 22. In some cases, a suitable FGF is FGF4, FGF5,and/or FGF6. In some cases, a suitable FGF is FGF4.

The amino acid sequences of exemplary human FGFs are:

FGF1: (SEQ ID NO: 2) MAEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRGPRTHYG QKAILFLPLPVSSD FGF2:(SEQ ID NO: 3) MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFL PMSAKS FGF3:(SEQ ID NO: 4) MGLIWLLLLSLLEPGWPAAGPGARLRRDAGGRGGVYEHLGGAPRRRKLYCATKYHLQLHPSGRVNGSLENSAYSILEITAVEVGIVAIRGLFSGRYLAMNKRGRLYASEHYSAECEFVERIHELGYNTYASRLYRTVSSTPGARRQPSAERLWYVSVNGKGRPRRGFKTRRTQKSSLFLPRVLDHRDHEMVRQLQSGLPRPPGKGVQPRRRRQKQSPDNLEPSHVQASRLGSQLE ASAH FGF4:(SEQ ID NO: 5) MSGPGTAAVALLPAVLLALLAPWAGRGGAAAPTAPNGTLEAELERRWESLVALSLARLPVAAQPKEAAVQSGAGDYLLGIKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSLLELSPVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGMFIALSKNGKTK KGNRVSPTMKVTHFLPRL FGF5:(SEQ ID NO: 6) MSLSFLLLLFFSHLILSAWAHGEKRLAPKGQPGPAATDRNPRGSSSRQSSSSAMSSSSASSSPAASLGSQGSGLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVNGSHEANMLSVLEIFAVSQGIVGIRGVFSNKFLAMSKKGKLHASAKFTDDCKFRERFQENSYNTYASAIHRTEKTGREWYVALNKRGKAKRGCSPRVKPQHISTHFLPRFKQSEQPELSFTVTVPEKKKPPSPIKPKIPLSAPRKNTNSVKYRLKFRFG FGF6: (SEQ ID NO: 7)MALGQKLFITMSRGAGRLQGTLWALVFLGILVGMVVPSPAGTRANNTLLDSRGWGTLLSRSRAGLAGEIAGVNWESGYLVGIKRQRRLYCNVGIGFHLQVLPDGRISGTHEENPYSLLEISTVERGVVSLFGVRSALFVAMNSKGRLYATPSFQEECKFRETLLPNNYNAYESDLYQGTYIALSKYGR VKRGSKVSPIMTVTHFLPRIFGF7: (SEQ ID NO: 8) MHKWILTWILPTLLYRSCFHIICLVGTISLACNDMTPEQMATNVNCSSPERHTRSYDYMEGGDIRVRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLAMNKEGKLYAKKECNEDCNFKELILENHYNTYASAKWTHNGGEMFVALNQKGIPVRGKKTKKEQKTAHF LPMAIT FGF8:(SEQ ID NO: 9) MGSPRSALSCLLLHLLVLCLQAQEGPGRGPALGRELASLFRAGREPQGVSQQHVREQSLVTDQLSRRLIRTYQLYSRTSGKHVQVLANKRINAMAEDGDPFAKLIVETDTFGSRVRVRGAETGLYICMNKKGKLIAKSNGKGKDCVFTEIVLENNYTALQNAKYEGWYMAFTRKGRPRKGSKTRQHQREVHFMKRLPRGHHTTEQSLRFEFLNYPPFTRSLRGSQRTWAPEPR FGF9: (SEQ ID NO: 10)MAPLGEVGNYFGVQDAVPFGNVPVLPVDSPVLLSDHLGQSEAGGLPRGPAVTDLDHLKGILRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVDSGLYLGMNEKGELYGSEKLTQECVFREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGTRTKRHQKFTHFL PRPVDPDKVPELYKDILSQSFGF10: (SEQ ID NO: 11) MWKWILTHCASAFPHLPGCCCCCFLLLFLVSSVPVTCQALGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRR GQKTRRKNTSAHFLPMVVHSFGF16: (SEQ ID NO: 12) MAEVGGVFASLDWDLHGFSSSLGNVPLADSPGFLNERLGQIEGKLQRGSPTDFAHLKGILRRRQLYCRTGFHLEIFPNGTVHGTRHDHSRFGILEFISLAVGLISIRGVDSGLYLGMNERGELYGSKKLTRECVFREQFEENWYNTYASTLYKHSDSERQYYVALNKDGSPREGYRTKRHQKFTHFLP RPVDPSKLPSMSRDLFHYRFGF17: (SEQ ID NO: 13) MGAARLLPNLTLCLQLLILCCQTQGENHPSPNFNQYVRDQGAMTDQLSRRQIREYQLYSRTSGKHVQVTGRRISATAEDGNKFAKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTKRTRRPQPLT FGF18: (SEQ ID NO: 14)MYSAPSACTCLCLHFLLLCFQVQVLVAEENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKP FKYTTVTKRSRRIRPTHPAFGF20: (SEQ ID NO: 15) MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARSKRHQKFT HFLPRPVDPERVPELYKDLLMYTFGF22: (SEQ ID NO: 16) MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGRVQGTRWRHGQDSILEIRSVHVGVVVIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIEENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVSIn some embodiments, a subject stage 1 medium comprises an FGF (e.g.,FGF4, FGF5, FGF6, etc.) at a concentration in a range of from 5 ng/ml to50 ng/ml (e.g., from 5 ng/ml to 40 ng/ml, from 10 ng/ml to 40 ng/ml,from 10 ng/ml to 30 ng/ml, from 15 ng/ml to 25 ng/ml, from 17.5 ng/ml to22.5 ng/ml, 15 ng/ml, 17.5 ng/ml, 20 ng/ml, 22.5 ng/ml, 25 ng/ml or 30ng/ml).

Wnt signaling agonist—In some embodiments, a suitable “Stage 1 medium”includes a Wnt signaling agonist. A Wnt signaling agonist triggers thenuclear localization of β-Catenin and results in the activation ofcanonical Wnt signaling. There are two main branches of the Wntsignaling pathway: (1) the canonical β-Catenin dependent Wnt signalingpathway and (2) the non-canonical β-Catenin independent pathways, whichinclude planar cell polarity (PCP) signaling as well as Calciumsignaling (Gao, et. al, Cell Signal. 2010 May; 22(5):717-27. Epub 2009Dec. 13). As used herein, the terms “Wnt signaling” and “Wnt/β-Cateninsignaling” are used interchangeably to refer to the canonical β-Catenindependent Wnt signaling pathway. As such, a “Wnt signaling agonist”increases output from the β-Catenin dependent Wnt signaling pathway.Activation of the Wnt pathway culminates when the protein β-Cateninenters the cell nucleus (for recent review of the canonical β-Catenindependent Wnt signaling pathway see Clevers et. al., Cell. 2012 Jun. 8;149(6):1192-205: Wnt/β-catenin signaling and disease). However, in theabsence of Wnt signaling, free cytosolic β-Catenin is incorporated intoa complex, known in the art as the β-Catenin destruction complex, whichincludes the proteins Axin, Adenomatous Polyposis Coli (APC), andglycogen synthase kinase (GSK-3β). Phosphorylation of β-Catenin byGSK-3β designates β-Catenin for the ubiquitin pathway and degradation byproteasomes (via βTRCP).

The binding of a canonical Wnt to its receptor (a Frizzled protein)leads to the activation of Dishevelled (Dvl) proteins, which inhibitglycogen synthase kinase-3β (GSK-3β) activity (i.e., phosphorylation ofβ-Catenin), leading to the cytosolic stabilization of β-Catenin.Stabilized β-Catenin then enters the nucleus and associates with theTCF/LEF (T Cell-specific transcription Factor/Lymphoid Enhancer Factor)family of transcription factors to induce transcription of importantdownstream target genes. Thus, in the absence of Wnt signaling,cytosolic (and therefore nuclear) levels of β-Catenin are kept low bynegative regulatory components of the pathway while in the presence ofWnt signaling, cytosolic (and therefore nuclear) levels of β-Catenin arestabilized by positive regulatory components of the pathway.

By “negative regulatory components” of the Wnt pathway, it is meantproteins that function by antagonizing the Wnt pathway, thus resultingin decreased pathway output (i.e., decreased target gene expression).Examples of known negative regulatory components of the Wnt pathwayinclude, but are in no way limited to: WIF, sFRP, Dkk, APCDD1, Notum,SOST, Axin, APC, GSK-3β, CK1γ, WTX, and βTrCP.

By “positive regulatory components” of the Wnt pathway, it is meantproteins that function by enhancing the Wnt pathway, thus resulting inincreased pathway output (i.e., increased target gene expression).Examples of known positive regulatory components of the Wnt pathwayinclude, but are in no way limited to: Wnt (e.g., Wnt1, Wnt3, Wnt3a,Wnt7a, and/or Wnt8), Norrin, R-spondin, PORCN, Wls, Frizzled, LRPS andLRP6, Tspan12, Lgr4, Lgr5, Lgr6, Dvl, β-Catenin, and TCF/LEF. In someembodiments, a subject Wnt signaling agonist is a positive regulatorycomponent of the canonical Wnt signaling pathway (e.g., Wnt1, Wnt3,Wnt3a, Wnt7a, Wnt8, etc.).

In some embodiments, a subject Wnt signaling agonist is “specific for”or “specifically binds to” a component of the Wnt signaling pathway suchthat binding results in increased pathway output (i.e., transcription oftarget genes). The binding of an agonist can be mediated by covalent ornon-covalent interactions or a combination of covalent and non-covalentinteractions. When the interaction of the agonist with the component towhich it specifically binds produces a non-covalently bound complex, thebinding which occurs is typically electrostatic, hydrogen-bonding, orthe result of lipophilic interactions. In particular, specific bindingis characterized by the preferential binding of one member of a pair toa particular species relative to other species within the family ofcompounds to which the corresponding member of the binding memberbelongs. Thus, for example, a Wnt signaling agonist that is specific forthe negative regulatory component GSK-3 preferably binds to GSK-3relative to other proteins in the cell.

A subject Wnt signaling agonist is any molecule (e.g., a chemicalcompound; a non-coding nucleic acid, e.g., a non-coding RNA; apolypeptide; a nucleic acid encoding a polypeptide, etc.) that resultsin increased output (i.e., increased target gene expression) from theWnt signaling pathway. For example, a Wnt signaling agonist can functionby stabilizing, enhancing the expression of, or enhancing the functionof a positive regulatory component of the pathway or by destabilizing,decreasing the expression of, or inhibiting the function of a negativeregulatory component of the pathway. Thus, a Wnt signaling agonist canbe a polypeptide positive regulatory component (e.g., Wnt3, wnt3a, Wnt1,and the like), and/or a nucleic acid encoding one or more positiveregulatory components of the pathway. A Wnt signaling agonist can alsobe a small molecule or nucleic acid that stabilizes a positiveregulatory component of the pathway either at the level of mRNA orprotein.

In some embodiments, a Wnt signaling agonist functions by stabilizingβ-Catenin, thus allowing nuclear levels of β-Catenin to rise. β-Catenincan be stabilized in multiple different ways. As multiple differentnegative regulatory components of the Wnt signaling pathway function byfacilitating the degradation of β-Catenin, a subject Wnt signalingagonist can be a small molecule or nucleic acid inhibitor (e.g.,microRNA, shRNA, etc.)(functioning at the level of mRNA or protein) of anegative regulatory component of the pathway. For example, in someembodiments, the Wnt signaling agonist is an inhibitor of GSK-3β. Insome such embodiments, the inhibitor of GSK-3β is a small moleculechemical compound (e.g., TWS119, BIO, CHIR-99021, SB 216763, SB 415286,CHIR-98014 and the like).

-   TWS119:    3-(6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)phenol (Ding    et. al, Proc Natl Acad Sci USA. 2003 Jun. 24; 100(13):7632-7. Epub    2003 Jun. 6)-   BIO:    6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihydro-(3Z)-2H-indol-2-one-   or (2′Z,3′E)-6-Bromoindirubin-3′-oxime-   (Meijer et. al, Chem Biol. 2003 Dec.; 10(12):1255-66)-   CHIR-99021:    6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile-   (Bennett et al., J Biol Chem. 2002 Aug. 23; 277(34):30998-1004. Epub    2002 Jun. 7)-   SB 216763:    3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione-   (Cross et al., J Neurochem. 2001 April; 77(1):94-102)-   SB 415286:    3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione-   (Cross et al., J Neurochem. 2001 April; 77(1):94-102)-   CHIR-98014:    N2-(2-(4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-ylamino)ethyl)-5    nitropyridine-2,6-diamine-   (Ring et al., Diabetes. 2003 March; 52(3):588-95)

Any convenient Wnt agonist may be used. In some embodiments, the Wntagonist is Wnt3a. The amino acid sequence of human Wnt3a is:

(SEQ ID NO: 17) MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRC VFHWCCYVSCQECTRVYDVHTCK.In some cases, a subject stage 1 medium comprises Wnt3a at aconcentration in a range of from about 20 ng/ml to about 80 ng/ml (e.g.,from about 20 ng/ml to about 80 ng/ml, from about 25 ng/ml to about 75ng/ml, from about 30 ng/ml to about 70 ng/ml, from about 35 ng/ml toabout 65 ng/ml, from about 40 ng/ml to about 60 ng/ml, from about 45ng/ml to about 55 ng/ml, from about 47.5 ng/ml to about 52.5 ng/ml, orabout 50 ng/ml).

Stage 2 medium. “Stage 2 medium” is also referred to herein as a “secondculture medium.” A suitable stage 2 medium is a basal culture medium(e.g., hepatocyte culture medium (HCM™), advanced RPMI 1640, RPMI 1640,etc.) suitable for the culture of cells (e.g., differentiating ASCs,hepatocytes, etc.) supplemented with at least hepatocyte growth factor(HGF). In some cases, stage 2 medium includes a fibroblast growth factor(e.g., FGF4, e.g., 25 ng/ml).

In some embodiments, a stage 2 media further includes a componentselected from: an FGF (e.g., FGF4, e.g., 25 ng/ml), oncostatinM (OSM,e.g., 30 ng/ml), dexamethasone (Dex, e.g., 2×10⁻⁵ M), dimethyl sulfoxide(DMSO, e.g., 0.1%), and a combination thereof. In some cases, the basalculture media used for stage 2 media is CLONETICS™ (available, forexample, from Lonza—Catalog number 3198) and it is supplemented with HGF(e.g., 150 ng/ml), an FGF (e.g., FGF4, e.g., 25 ng/ml), oncostatinM(e.g., 30 ng/ml), dexamethasone (e.g., 2×10⁻⁵ M), and DMSO (e.g., 0.1%).

Hepatocyte growth factor (HGF) is a protein that functions as a potentmitogen, hepatotrophic factor, and/or a growth factor. The amino acidsequence of human HGF is:

(SEQ ID NO: 18) MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGI FVRVAYYAKWIHKIILTYKVPQS.In some embodiments, a subject stage 2 medium includes HGF at aconcentration in a range of from 75 ng/ml to 250 ng/ml (e.g., from 100ng/ml to 200 ng/ml, from 120 ng/ml to 180 ng/ml, from 125 ng/ml to 175ng/ml, from 130 ng/ml to 170 ng/ml, from 135 ng/ml to 165 ng/ml, from140 ng/ml to 160 ng/ml, from 145 ng/ml to 155 ng/ml, 120 ng/ml, 125ng/ml, 130 ng/ml, 135 ng/ml, 140 ng/ml, 145 ng/ml, 150 ng/ml, 155 ng/ml,160 ng/ml or 175 ng/ml).

In some embodiments, a subject stage 2 medium includes an FGF (e.g.,FGF4) at a concentration in a range of from 5 ng/ml to 55 ng/ml (e.g.,from 5 ng/ml to 45 ng/ml, from 10 ng/ml to 45 ng/ml, from 10 ng/ml to 35ng/ml, from 15 ng/ml to 35 ng/ml, from 20 ng/ml to 30 ng/ml, from 22.5ng/ml to 27.5 ng/ml, 15 ng/ml, 17.5 ng/ml, 20 ng/ml, 22.5 ng/ml, 25ng/ml, 27.5 ng/ml, or 30 ng/ml).

Oncostatin M (OSM) is a polypeptide that functions as a cytokine andbelongs to the interleukin 6 group of cytokines. The amino acid sequenceof human OSM is:

(SEQ ID NO: 19) MGVLLTQRTLLSLVLALLFPSMASMAAIGSCSKEYRVLLGQLQKQTDLMQDTSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEETLRGLGRRGFLQTLNATLGCVLHRLADLEQRLPKAQDLERSGLNIEDLEKLQMARPNILGLRNNIYCMAQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKLEGCRFLHGYHRFMHSVGRVFSKWGESPNRSRRHSPHQALRKGVRRT RPSRKGKRLMTRGQLPRIn some embodiments, a subject stage 2 medium includes oncostatinM (OSM)at a concentration in a range of from 10 ng/ml to 50 ng/ml (e.g., from15 ng/ml to 45 ng/ml, from 20 ng/ml to 40 ng/ml, from 22.5 ng/ml to 37.5ng/ml, from 25 ng/ml to 35 ng/ml, from 27.5 ng/ml to 32.5 ng/ml, 25ng/ml, 27.5 ng/ml, 30 ng/ml, 32.5 ng/ml, or 35 ng/ml).

Dexamethasone (Dex) is a synthetic member of the glucocorticoid class ofsteroid drugs that has anti-inflammatory and immunosuppressantproperties. Dex is also known as(11β,16α)-9-Fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione,9α-Fluoro-16α-methyl-11β,17β,21-trihydroxy-1,4-pregnadiene-3,20-dione,9α-Fluoro-16α-methylprednisolone, and Prednisolone F. Dex has the CASnumber 50-02-2 and the chemical structure:

In some embodiments, a subject stage 2 medium comprises dexamethasone(Dex) at a concentration in a range of from 1×10⁻⁶ M to 1×10⁻⁴ M (e.g.,from 5×10⁻⁶ to 5×10⁻⁵ M, from 1×10⁻⁵ M to 3×10⁻⁵ M, from 1.5×10⁻⁵ M to2.5×10⁻⁵ M, or 2×10⁻⁵ M).

Dimethylsulfoxide (DMSO) is an organosulfur compound with the formula(CH₃)₂SO that is a colorless liquid and is a polar aprotic solvent thatdissolves both polar and nonpolar compounds. DMSO is also known asMethyl sulfoxide, has the CAS number 67-68-5, and has the chemicalstructure:

In some embodiments, a subject stage 2 medium comprises DMSO at aconcentration in a range of from 0.01% to 1% (e.g., from 0.025% to 0.5%,from 0.05% to 0.2%, or 0.1%).

Hepatocyte-like cells. The term “hepatocyte-like cell” as used hereinrefers to a cell (e.g., of a cell population, e.g., a cell that isinduced according to the subject methods) that is positive for (i.e.,exhibits) one or more hepatocyte characters. Hepatocyte characters(i.e., characteristics of hepatocyte-like cells) include, but are notlimited to:

-   (i) expression of (i.e., positive for) one or more hepatocyte    markers (e.g., glucose-6-phosphatase, albumin (ALB),    alpha-1-antitrypsin (AAT, also known as SERPINA1), cytokeratin 8    (CK8), cytokeratin 18 (CK18), cytokeratin 8/18 (CK8/18),    asialoglycoprotein receptor 1 (ASGR1), alcohol dehydrogenase 1,    arginase Type I, cytochrome p450 3A4 (CYP3A4), liver-specific    organic anion transporter (LST-1), forkhead box protein A2 (FoxA2),    alpha-fetoprotein (AFP), tryptophan 2,3-dioxygenase (TDO2), or a    combination thereof);-   (ii) activity of liver enzymes such as glucose-6-phosphatase,    CYP3A4, and/or CYP1A1;-   (iii) production and/or secretion of liver products (e.g., as    measured in bodily fluids such as blood, serum, plasma, etc.)(e.g.,    bile, urea, and/or albumin);-   (iv) exhibition of a hepatocyte metabolic properties (e.g., ability    to detoxify xenobiotics; endocytosis of LDL, synthesis of glycogen,    cytochrome P450 1A2 detoxification activity, and the like);-   (v) exhibition of hepatocyte morphological features;-   (vi) ability to engraft into the liver of an immunodeficient    indidual (e.g., a mouse, a human, etc.); and-   (vii) lack of expression of (negative for) one or more    non-hepatocyte markers (e.g., adipocyte markers, e.g., CD37, CD29,    etc.; ASC markers, e.g., CD105; and the like).

A subject hepatocyte-like cell does not have to be positive for alltested hepatocyte characters in order for the cell to be considered ahepatocyte-like cell. For example, in some cases, a subjecthepatocyte-like cell is positive for all tested hepatocyte characters.However, in some cases, a subject hepatocyte-like cell is positive forone or more hepatocyte character; and is also negative for one or morehepatocyte characters. In some cases (described below in more detail), ahepatocyte-like cell is transplanted into an individual. In such cases,the hepatocyte-like cell can be (i) positive for all tested hepatocytecharacters; or (ii) positive for one or more hepatocyte character(s);and negative for one or more hepatocyte character(s).

Thus, in some cases, hepatocyte-like cells do not express all knownhepatocyte markers and/or test positive for all hepatocyte functionaltests. For example, in some cases, hepatocyte-like cells that areinduced according to the subject methods do not express ASGR1. However,as demonstrated in the examples section below (e.g., FIG. 3C), in somecases, hepatocyte-like cells exhibit a hepatocyte character (e.g.,express a hepatocyte marker such as ASGR1) after transplantation (e.g.,into an individual such as a mouse and/or a human) that they did notexhibit prior to transplantation. For example, in some cases,hepatocyte-like cells that are induced according to the subject methodsdo not express the hepatocyte marker ASGR1 prior to transplantation intoan individual, but do express ASGR1 after transplantation. Thus, in somecases, it is not required that hepatocyte-like cells induced accordingto the subject methods exhibit all known hepatocyte characters (e.g.,production of a liver product, expression of a hepatocyte marker, etc.)or even exhibit all tested hepatocyte characters prior to the use of thecells for transplantation. For example, in some cases, hepatocyte-likecells that are induced according to the subject methods are positive forone or more hepatocyte characters, and are negative for one or morehepatocyte characters, but are still considered hepatocyte-like cellsand can still be transplanted into an individual.

In some embodiments, the subject methods include the step of verifyingthe presence of induced hepatocyte-like cells (iHeps) in the inducedcell population. Verifying can rely on cellular phenotypes (e.g., geneor protein expression, drug metabolism profile, responsiveness toparticular drugs, etc.) known in the art to be characteristic ofhepatocytes. For example, verifying can be performed by testing for anyone or more of the hepatocyte characters listed above (e.g., expressionof a hepatocyte marker, lack of a expression of a non-hepatocyte marker,production and/or secretion of a liver product, activity of a liverenzyme, exhibition of to hepatocyte metabolic property, etc.). In somecases, verifying comprises determining the percentage of cells of theinduced cell population that are iHeps.

In some embodiments, verifying includes contacting cells of the inducedcell population with a specific binding agent (e.g., a nucleic acidprobe, an antibody, etc.) specific for a hepatocyte marker (e.g., mRNA,protein, etc.), and determining the percentage of cells positive forexpression, wherein cells positive for expression are iHeps. Suitablemarkers are listed above. In some embodiments, verifying includescontacting the induced cell population with a binding agent (e.g., anucleic acid probe, an antibody, etc.) specific for a non-hepatocytemarker (e.g., mRNA, protein, etc.), and determining the percentage ofcells negative for expression, wherein cells negative for expression areiHeps. Suitable markers are listed above. In some cases, 10% or more ofthe cells of the induced cell population are determined to be iHeps(e.g., 10.5% or more, 11% or more, 12.5% or more, 15% or more, 17.5% ormore, 20% or more, 22.5% or more, 25% or more, 27.5% or more, 30% ormore, 32.5% or more, 35% or more, 37% or more, 40% or more, 45% or more,50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% ormore, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more,99% or more, or 100%). In some embodiments, the percent of cells of theinduced cell population that are determined to be iHeps is in a range offrom 10% to 100% (e.g., from 10% to 90%, from 10% to 80%, from 10% to70%, from 10% to 60%, from 10% to 50%, from 10% to 45%, from 10% to100%, from 40% to 100%, from 10% to 37.5%, from 10% to 37%, from 15% to90%, from 15% to 80%, from 15% to 70%, from 15% to 60%, from 15% to 50%,from 15% to 45%, from 15% to 100%, from 40% to 100%, from 15% to 37.5%,from 15% to 37%, from 20% to 90%, from 20% to 80%, from 20% to 70%, from20% to 60%, from 20% to 50%, from 20% to 45%, from 20% to 100%, from 40%to 100%, from 20% to 37.5%, from 20% to 37%, from 25% to 90%, from 25%to 80%, from 25% to 70%, from 25% to 60%, from 25% to 50%, from 25% to45%, from 25% to 100%, from 40% to 100%, from 25% to 37.5%, from 25% to37%, from 30% to 90%, from 30% to 80%, from 30% to 70%, from 30% to 60%,from 30% to 50%, from 30% to 45%, from 30% to 100%, from 40% to 100%,from 30% to 37.5%, or from 30% to 37%).

It will be understood by those of skill in the art that expressionlevels reflect detectable amounts of the marker (e.g., nucleic acid orprotein) on and/or in the cell. A cell that is negative for staining(e.g., the level of binding of a marker specific reagent is notdetectably different from a matched control) may still express minoramounts of the marker. And while it is commonplace in the art to referto cells as “positive” or “negative” for a particular marker, actualexpression levels are quantitative traits. The number of detectedmolecules can vary by several logs, yet still be characterized as“positive”.

When a protein marker is used, the staining intensity (e.g., of amarker-specific antibody) can be monitored by flow cytometry, wherelasers detect the quantitative levels of fluorochrome (which isproportional to the amount of cell marker bound by specific reagents,e.g. antibodies). Flow cytometry, or FACS, can also be used to separatecell populations based on the intensity of binding to a specificreagent, as well as other parameters such as cell size and lightscatter. Although the absolute level of staining may differ with aparticular fluorochrome and reagent preparation, the data can benormalized to a control.

In order to normalize the distribution to a control, each cell isrecorded as a data point having a particular intensity of staining.These data points may be displayed according to a log scale, where theunit of measure is arbitrary staining intensity. In one example, thebrightest stained cells in a sample can be as much as 4 logs moreintense than unstained cells. When displayed in this manner, it is clearthat the cells falling in the highest log of staining intensity arebright, while those in the lowest intensity are negative. The “low”positively stained cells have a level of staining brighter than that ofan isotype matched control, but is not as intense as the most brightlystaining cells normally found in the population. An alternative controlmay utilize a substrate having a defined density of marker on itssurface, for example a fabricated bead or cell line, which provides thepositive control for intensity.

Enrichment of hepatocyte-like cells. To increase the fraction ofobtained cells that are iHeps, it is sometimes advantageous to enrichfor (i.e., purify) the produced iHeps. In particular aspects, thehepatocyte-like cells provided herein may be selected or enriched byusing a screenable or selectable reporter expression cassette comprisinga mature hepatocyte-specific transcriptional regulatory element operablylinked to a reporter gene, or by cell sorting (e.g., magnetic cellsorting, Fluorescence Activated Cell Sorting (FACS), and the like) usingan antibody against a hepatocyte-specific marker (e.g., a cell surfaceantigen such as ASGR1).

To aid selection or enrichment, the ASCs, may comprise a selectable orscreenable reporter expression cassette comprising a reporter gene. Thereporter expression cassette may comprise a hepatocyte-specifictranscriptional regulatory element operably linked to a reporter gene(e.g., any of the fluorescent proteins, e.g., blue, yellow, red, green,etc.). Non-limiting examples of hepatocyte-specific transcriptionalregulatory element include a promoter of glucose-6-phosphatase, albumin(ALB), alpha-1-antitrypsin (AAT, also known as SERPINA1), cytokeratin 8(CK8), cytokeratin 18 (CK18), asialoglycoprotein receptor 1 (ASGR1),alcohol dehydrogenase 1, arginase Type I, cytochrome p450 3A4 (CYP3A4),liver-specific organic anion transporter (LST-1), forkhead box proteinA2 (FoxA2), alpha-fetoprotein (AFP), and tryptophan 2,3-dioxygenase(TDO2).

Enrichment using antibodies (e.g., magnetic cell sorting, FACS, and thelike) specific for cell surface markers of ASCs (e.g., CD29, CD34, CD36,CD49f, CD73, CD90 (Thy-1), CD105, CD133, c-kit, c-met, and the like),endodermal precursor cells (e.g., SOX17), hepatocyteprecursor/progenitor cells (e.g., N-cadherin, E-cadherin, EpCAM, andNCAM), and/or hepatocytes (e.g., ASGR1) have the advantage of notrequiring genetic modification of the cells to be enriched. Magneticcell sorting and FACS have the ability to analyze multiple surfacemarkers simultaneously, and they can be used to sort ASCs, endodermalprecursor cells, hepatocyte precursor/progenitor cells, and/orhepatocytes (e.g., iHeps) based on the expression of a cell surfacemarker. In some cases, iHeps do not express ASGR1. However, in somecases, e.g, if they are cultured longer and/or are furtherdifferentiated, iHeps do express ASGR1. In cases where cells that (i) donot express ASGR1; and/or (ii) express markers of hepatocyte precursorcells and/or endodermal precursor cells, are administered to anindividual, the cells can further differentiate after administrationinto the individual (as demonstrated in the examples section below).

In some cases, cells that are produced by the subject methods can beenriched (e.g., sorted) by using an expressed cell surface marker. Forexample, cells of the precursor cell population (e.g., ASCs that havebeen contacted by a stage 1 medium) and/or cells of the induced cellpopulation (e.g., cells of the precursor cell population that have beencontacted with stage 2 medium) can be enriched using any marker that isexpressed by hepatocyte precursor/progenitor cells (e.g., N-cadherin,E-cadherin, EpCAM, and NCAM) and/or endodermal precursor cells (e.g.,SOX17). Cells of the induced cell population (e.g., cells of theprecursor cell population that have been contacted with stage 2 medium)can be enriched using any marker that is expressed by hepatocyteprecursor/progenitor cells (e.g., N-cadherin, E-cadherin, EpCAM, NCAM,etc.), endodermal precursor cells (e.g., SOX17), and/or hepatocytes(e.g., ASGR1) prior to further culture (e.g., to facilitate for furtherdifferentiation) and/or prior to administration to an individual. A cellpopulation that includes ASCs (e.g., a population of cells fromliposuction) can be enriched for ASCs using ASC cell surface markers(e.g., CD29, CD34, CD36, CD49f, CD73, CD90 (Thy-1), CD105, CD133, c-kit,c-met, and the like).

Cryopreservation. In some embodiments, subject cells (e.g., iHeps) arepreserved for future use. Specifically, iHeps can be cryopreserved byperforming the following steps: (i) Detach cells from growth cellsurface, (ii) rinse cells (e.g., wash with PBS via centrifugation),(iii) resuspend cells (e.g., cell pellet) in media with 10% DMSO, and(vi) freeze cells and store in liquid nitrogen using standard tissueculture techniques commonly used in the art to preserve cells. Cells canbe frozen at 1-50 million cells per vial. When ready for use, iHepsshould be thawed in a manner as commonly known in the art for thawingfrozen cultured cells.

Methods of Treatment

Aspects of the disclosure include methods of treating an individual. Thesubject methods of treatment generally include producing a population ofhepatocyte-like cells from a population of adipose-derived stem cells(e.g., using the methods described above), and administering (e.g.,injecting, transplanting, etc.) an effective number of hepatocyte-likecells into an individual. The ASCs can be from any source and can bederived by any convenient method. Exemplary methods ofisolating/extracting/obtaining ASCs are described above. In some casesthe ASCs are autologous (i.e., from the same individual into which theiHeps will be administered) (e.g., to reduce the possibility and/orseverity of an immune response). In some cases, the ASCs are from arelated individual (e.g., to reduce the possibility and/or severity ofan immune response). In some cases, the ASCs are from an unrelatedindividual. In some cases, the ASCs are from an individual of anotherspecies (e.g., human ASCs administered to a mouse).

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom(s) thereofand/or may be therapeutic in terms of a partial or completestabilization or cure for a disease and/or adverse effect attributableto the disease. The term “treatment” encompasses any treatment of adisease in a mammal, particularly a human, and includes: (a) preventingthe disease and/or symptom(s) from occurring in a subject who may bepredisposed to the disease or symptom but has not yet been diagnosed ashaving it; (b) inhibiting the disease and/or symptom(s), i.e., arrestingdevelopment of a disease and/or the associated symptoms; or (c)relieving the disease and the associated symptom(s), i.e., causingregression of the disease and/or symptom(s). Those in need of treatmentcan include those already inflicted (e.g., those with liver malfunction,liver disease, liver damage, etc.) as well as those in which preventionis desired.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs,camels, etc. In some embodiments, the mammal is human.

A therapeutic treatment is one in which the subject is inflicted priorto administration and a prophylactic treatment is one in which thesubject is not inflicted prior to administration. In some embodiments,the subject has an increased likelihood of becoming inflicted or issuspected of being inflicted prior to treatment. In some embodiments,the subject is suspected of having an increased likelihood of becominginflicted.

In some embodiments, the individual to be treated is an individual withreduced liver function (e.g., an individual with liver damage). The term“liver damage” as used herein refers to any damage (e.g., caused bydisease, trauma, unexplained, etc.) resulting in reduced liver function.An individual with reduced liver function (e.g., reduced relative to theindividual's basal level of function; reduced relative to a healthyindividual of comparable age, weight, sex, etc.; and the like) isreferred to herein as an individual with a damaged liver (i.e., anindividual with liver damage). Thus, the terms “liver damage” and“reduced liver function” are used synonymously herein. In some cases, anindividual with “liver damage” is an individual with liver disease.

The cause for reduced liver function may be known or unknown. How todetermine whether a given individual has reduced liver function (andtherefore also how to determine whether an individual receivingtreatment is recovering or has recovered liver function) will be knownto one of ordinary skill in the art. Liver function tests can includeliver enzyme tests, also known as liver function tests (LFTs), a groupof blood tests that detect inflammation and damage to the liver. Bloodprotein markers of liver damage may include, for example: increasedaspartate aminotransferase (AST), also known as SGOT; increased alanineaminotransferase (ALT), also known as SGPT; increased alkalinephosphatase; increased 5′ nucleotidase; decreased albumin; decreasedglobulin; and increased gamma-glutamyl transpeptidase (GGT). If, forexample, elevated amounts (and/or activity) of the above protein markers(or a decreased level for albumin and/or globulin) are detected in theblood, liver damage may be present. Additional liver function testsinclude: (i) prothrombin time (PT), a test of the time it takes for ablood sample to clot (e.g., if low levels of clotting factors arepresent, the prothrombin time is longer), which can be reported asinternational normalized ratio (INR) (a standardized way forlaboratories to report PT so that results can be compared accuratelyacross laboratories); and (ii) a bilirubin test: bilirubin blood levelsmay be elevated in people with impaired bile flow, which can occur insevere liver disease, gallbladder disease, or other bile systemconditions.

Examples of symptoms, illnesses, and/or diseases that can be consideredto indicate “liver damage” (and that can be treated by transplantingsubject induced hepatocyte-like cells into an individual having thesymptoms, illness, and/or disease), include, but are not limited to:acute liver failure, alcohol-related liver disease, Alagille syndrome,alpha 1-antitrypsin deficiency, alveolar hydatid disease, autoimmunehepatitis, bacillary peliosis, biliary atresia, Budd-Chiari syndrome,chronic liver disease, cirrhosis, congenital hepatic fibrosis,congestive hepatopathy, fatty liver, galactosemia, gastric antralvascular ectasia, Gilbert syndrome, hemochromatosis, hepatitis (A, B,and/or C), hepatic encephalopathy, hepato-biliary diseases,hepatolithiasis, hepatopulmonary syndrome, hepatorenal syndrome,hepatosplenomegaly, hepatotoxicity, hepatocellular carcinoma, hepaticencephalopathy, jaundice, Laennec's cirrhosis, liver abscess, livercysts, liver cancer, liver failure, Lyngstadaas syndrome, non-alcoholicfatty liver disease, pediatric end-stage liver disease, peliosishepatis, polycystic liver disease, primary biliary cirrhosis, primarysclerosing cholangitis (PSC), progressive familial intrahepaticcholestasis, Reye syndrome, type I glycogen storage disease, viralhepatitis, Wilson disease, Zahn infarct, and Zieve's syndrome.

In some cases, iHeps are cultured for a period of time prior totransplantation (e.g., in HCM™ for 2 days). Cells (e.g., iHeps) can beprovided to the individual (i.e., administered into the individual)alone or with a suitable substrate or matrix, e.g. to support theirgrowth and/or organization in the tissue to which they are beingtransplanted (e.g., liver). In some embodiments, the matrix is ascaffold (e.g., an organ scaffold). In some embodiments, 1×10³ or morecells will be administered (e.g., transplanted), for example 5×10³ ormore cells, 1×10⁴ or more cells, 5×10⁴ or more cells, 1×10⁶ or morecells, 5×10⁵ or more cells, 1×10⁶ or more cells, 5×10⁶ or more cells,1×10⁷ or more cells, 5×10⁷ or more cells, 1×10⁸ or more cells, 5×10⁸ ormore cells, 1×10⁹ or more cells, 5×10⁹ or more cells, or 1×10¹⁰ or morecells. In some embodiments, subject cells are administered into theindividual on microcarriers (e.g., cells grown on biodegradablemicrocarriers).

The cells induced by the subject methods (iHeps) may be administered inany physiologically acceptable excipient (e.g., William's E medium),where the cells may find an appropriate site for survival and function(e.g., organ reconstitution). The cells may be introduced by anyconvenient method (e.g., injection, catheter, or the like).

The cells may be introduced to the subject (i.e., administered into theindividual) via any of the following routes: parenteral, subcutaneous,intravenous, intracranial, intraspinal, intraocular, or into spinalfluid. The cells may be introduced by injection (e.g., direct localinjection), catheter, or the like. Examples of methods for localdelivery (e.g., delivery to the liver) include, e.g., by bolusinjection, e.g. by a syringe, e.g. into a joint or organ; e.g., bycontinuous infusion, e.g. by cannulation, e.g. with convection (see e.g.US Application No. 20070254842, incorporated here by reference); or byimplanting a device upon which the cells have been reversably affixed(see e.g. US Application Nos. 20080081064 and 20090196903, incorporatedherein by reference).

In some cases, iHeps are administered into an individual byultrasound-guided liver injection. In this way, cells can be placeddirectly into a liver lobe (e.g., in humans, or even in mice using asmall animal ultrasound system). Brightness mode (B-mode) can be used toacquire two-dimensional images for an area of interest with a transducerand cells can be injected in solution (e.g., 100 μl to 300 μl, e.g., 200μl of, for example, William's E medium) into one site or many sites(e.g., 1-30 sites) in the liver using, for example, a 30G needle.

The number of administrations of treatment to a subject may vary.Introducing cells into an individual may be a one-time event; but incertain situations, such treatment may elicit improvement for a limitedperiod of time and require an on-going series of repeated treatments. Inother situations, multiple administrations of iHeps may be requiredbefore an effect is observed. As will be readily understood by one ofordinary skill in the art, the exact protocols depend upon the diseaseor condition, the stage of the disease and parameters of the individualbeing treated.

A “therapeutically effective dose” or “therapeutic dose” is an amountsufficient to effect desired clinical results (i.e., achieve therapeuticefficacy). A therapeutically effective dose can be administered in oneor more administrations. For purposes of this disclosure, atherapeutically effective dose of iHeps is an amount that is sufficient,when administered to (e.g., transplanted into) the individual, topalliate, ameliorate, stabilize, reverse, prevent, slow or delay theprogression of the disease state (e.g., liver damage) by, for example,providing functions normally provided by a healthy liver. In some cases,transplanted iHeps integrate into the individual's liver and become partof the liver. In some cases, transplanted iHeps do not integrate intothe individual's liver, but can still provide functions normallyprovided by a healthy liver.

In some embodiments, a therapeutically effective dose of iHeps is about1×10³ or more cells (e.g., 5×10³ or more, 1×10⁴ cells, 5×10⁴ or more,1×10⁵ or more, 5×10⁵ or more, 1×10⁶ or more, 5×10⁶ or more, 1×10⁷ cells,5×10⁷ or more, 1×10⁸ or more, 5×10⁸ or more, 1×10⁹ or more, 5×10⁹ ormore, or 1×10¹⁰ or more). In some embodiments, a therapeuticallyeffective dose of iHeps is in a range of from about 1×10³ cells to about1×10¹⁰ cells (e.g, from about 5×10³ cells to about 1×10¹⁰ cells, fromabout 1×10⁴ cells to about 1×10¹⁰ cells, from about 5×10⁴ cells to about1×10¹⁰ cells, from about 1×10⁵ cells to about 1×10¹⁰ cells, from about5×10⁵ cells to about 1×10¹⁰ cells, from about 1×10⁶ cells to about1×10¹⁰ cells, from about 5×10⁶ cells to about 1×10¹⁰ cells, from about1×10⁷ cells to about 1×10¹⁰ cells, from about 5×10⁷ cells to about1×10¹⁰ cells, from about 1×10⁸ cells to about 1×10¹⁰ cells, from about5×10⁹ cells to about 1×10¹⁰, from about 5×10³ cells to about 5×10⁹cells, from about 1×10⁴ cells to about 5×10⁹ cells, from about 5×10⁴cells to about 5×10⁹ cells, from about 1×10⁵ cells to about 5×10⁹ cells,from about 5×10⁵ cells to about 5×10⁹ cells, from about 1×10⁶ cells toabout 5×10⁹ cells, from about 5×10⁶ cells to about 5×10⁹ cells, fromabout 1×10⁷ cells to about 5×10⁹ cells, from about 5×10⁷ cells to about5×10⁹ cells, from about 1×10⁸ cells to about 5×10⁹ cells, from about5×10⁸ cells to about 5×10⁹, from about 5×10³ cells to about 1×10⁹ cells,from about 1×10⁴ cells to about 1×10⁹ cells, from about 5×10⁴ cells toabout 1×10⁹ cells, from about 1×10⁵ cells to about 1×10⁹ cells, fromabout 5×10⁵ cells to about 1×10⁹ cells, from about 1×10⁶ cells to about1×10⁹ cells, from about 5×10⁶ cells to about 1×10⁹ cells, from about1×10⁷ cells to about 1×10⁹ cells, from about 5×10⁷ cells to about 1×10⁹cells, from about 1×10⁸ cells to about 1×10⁹ cells, from about 5×10⁸cells to about 1×10⁹, from about 5×10³ cells to about 5×10⁸ cells, fromabout 1×10⁴ cells to about 5×10⁸ cells, from about 5×10⁴ cells to about5×10⁸ cells, from about 1×10⁵ cells to about 5×10⁸ cells, from about5×10⁵ cells to about 5×10⁸ cells, from about 1×10⁶ cells to about 5×10⁸cells, from about 5×10⁶ cells to about 5×10⁸ cells, from about 1×10⁷cells to about 5×10⁸ cells, from about 5×10⁷ cells to about 5×10⁸ cells,or from about 1×10⁸ cells to about 5×10⁸ cells).

The cells of this disclosure can be supplied in the form of apharmaceutical composition, comprising an isotonic excipient preparedunder sufficiently sterile conditions for human administration. Forgeneral principles in medicinal formulation, the reader is referred toCell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister& P. Law, Churchill Livingstone, 2000. Choice of the cellular excipientand any accompanying elements of the composition will be adapted inaccordance with the route and device used for administration. Thecomposition may also comprise or be accompanied with one or more otheringredients that facilitate the engraftment or functional mobilizationof the cells. Suitable ingredients include matrix proteins that supportor promote adhesion of the cells, or complementary cell types.

Cells of the subject methods may be genetically altered in order tointroduce genes useful in the differentiated hepatocytes, e.g. repair ofa genetic defect in an individual, selectable marker, etc. Cells mayalso be genetically modified to enhance survival, control proliferation,and the like. Cells may be genetically altered by transfection ortransduction with a suitable vector, homologous recombination, or otherappropriate technique, so that they express a gene of interest. In someembodiments, a selectable marker is introduced, to provide for greaterpurity of the desired differentiating cell.

The cells of this disclosure can also be genetically altered in order toenhance their ability to be involved in tissue regeneration, or todeliver a therapeutic gene to a site of administration. A vector isdesigned using the known encoding sequence for the desired gene,operatively linked to a promoter that is either pan-specific orspecifically active in hepatocytes.

Many vectors useful for transferring exogenous genes into targetmammalian cells are available. The vectors may be episomal, e.g.plasmids, virus derived vectors such cytomegalovirus, adenovirus, etc.,or may be integrated into the target cell genome, through homologousrecombination or random integration, e.g. retrovirus derived vectorssuch MMLV, HIV-1, ALV, etc. For modification of stem cells, lentiviralvectors are preferred. Lentiviral vectors such as those based on HIV orFIV gag sequences can be used to transfect non-dividing cells, such asthe resting phase of human stem cells (see Uchida et al. (1998) P.N.A.S.95(20):11939-44).

Combinations of retroviruses and an appropriate packaging line may alsofind use, where the capsid proteins will be functional for infecting thetarget cells. Usually, the cells and virus will be incubated for atleast about 24 hours in the culture medium. The cells are then allowedto grow in the culture medium for short intervals in some applications,e.g. 24-73 hours, or for at least two weeks, and may be allowed to growfor five weeks or more, before analysis. Commonly used retroviralvectors are “defective”, i.e. unable to produce viral proteins requiredfor productive infection. Replication of the vector requires growth inthe packaging cell line.

The host cell specificity of the retrovirus is determined by theenvelope protein, env (p120). The envelope protein is provided by thepackaging cell line. Envelope proteins are of at least three types,ecotropic, amphotropic and xenotropic. Retroviruses packaged withecotropic envelope protein, e.g. MMLV, are capable of infecting mostmurine and rat cell types. Ecotropic packaging cell lines include BOSC23(Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearingamphotropic envelope protein, e.g. 4070A (Danos et al, supra.), arecapable of infecting most mammalian cell types, including human, dog andmouse. Amphotropic packaging cell lines include PA12 (Miller et al.(1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol.Cell. Biol. 6:2895-2902) GRIP (Danos et al. (1988) PNAS 85:6460-6464).Retroviruses packaged with xenotropic envelope protein, e.g. AKR env,are capable of infecting most mammalian cell types, except murine cells.

The vectors may include genes that must later be removed, e.g. using arecombinase system such as Cre/Lox, or the cells that express themdestroyed, e.g. by including genes that allow selective toxicity such asherpesvirus TK, bcl-xs, etc. Suitable inducible promoters are activatedin a desired target cell type, either the transfected cell, or progenythereof. By transcriptional activation, it is intended thattranscription will be increased above basal levels in the target cell byat least about 100 fold, more usually by at least about 1000 fold.

Utility

The results described in the Examples below demonstrate that functioninghuman liver tissue can be generated in vivo after direct implantation ofhepatocyte-like cells, derived from ASCs using the subject methods, intothe liver. The SCi-Heps (iHeps induced using the subject methods) wereproduced from ASCs by in vitro differentiation for a short period inchemically defined media. SCi-Heps exhibited many of the in vitrofunctional properties of mature hepatocytes, and they were able tostably reconstitute functioning human liver in vivo in a murine modelsystem, and implantation studies demonstrated that SCi-Heps have a verylow malignant potential. Thus, readily-accessible stem cells were usedto rapidly build functioning human liver tissue in vivo. The time togenerate iHeps from ASCs is greatly reduced by the subject methods tounder 13 days (e.g., 9 days). In addition, the efficiency of iHepproduction is greatly increased by the subject methods to greater that10% (e.g., 37%). Both of these features facilitate treatment methodsbecause liver damage (e.g., liver failure, e.g., due to acetaminophentoxicity) can rapidly cause death (e.g., in 2 weeks or less).

These procedures can be scaled to enable autologous liver regenerationin humans. Since there are ˜140×10⁶ hepatocytes per gram of human ormouse liver tissue; a 1500 gram adult human liver has ˜2×10¹¹hepatocytes, while a 1.8 gram mouse liver has 2.5×10⁶ cells. After 1million human hepatocytes are transplanted into a TK-NOG mouse, ˜50%replacement of the mouse hepatocytes is routinely obtained, whichamounts to ˜1.2×10⁶ functioning human hepatocytes in the mouse liver.This represents a 120-fold increase (or 7 population doublings) in thenumber of human hepatocytes generated in vivo relative to the number oftransplanted cells. If a similar regeneration efficiency is achieved inhumans, 800 million SCi-Heps (10¹¹/120) would be transplanted to replace50% of the cells in a human liver. Of relevance to this question, 1liter (1000 g) of lipo-aspirate can easily be obtained from a singleliposuction procedure. This volume is estimated to contain 2-10×10⁹cells, of which 1-10% are estimated to be ASCs. Thus, one liter oflipo-aspirate contains between 2×10⁷ and 10⁹ ASCs, which can provide asufficient number of cells to enable autologous human liver replacement.Human liver regenerative procedures, including ultrasound-guided directliver Implantation (used in the examples below), are scalable andappropriate for human clinical use.

The examples below demonstrate that spherical culture coupled with achemical differentiation process can increase the yield of SCi-Heps by7-fold compared to other known methods. Thus, the spherical culturemethod helps to ensure that a sufficient number of SCi-Heps can beobtained to enable liver regeneration in a human subject. The sphericalculture method also reduces the culture volume by 12-fold and decreasesthe time in culture by 40%; these factors dramatically reduce the costof differentiating ASC into iHeps. The estimated cost of the medium andgrowth factors required for producing Chi-Heps is ˜$49,000 per liter oflipo-aspirate processed, but the subject method reduces these costs byover 20-fold (to ˜$2400 per liter of lipo-aspirate processed).

The fact that implanted SCi-Heps did not form tumors (see the Examplesbelow) indicates that these cells have a markedly reduced risk ofcausing tumors. In contrast to the results obtained with SCi-Heps,readily palpable tumors were noted within 3 weeks after implantation ofiPS-Heps. The rapid tumor onset should not be surprising, since thisiPS-derived cell population has a large percentage of un-differentiatedcells. However, ˜10⁹ cells must be transplanted to regenerate a humanliver, but even as few as 100-1000 cells with malignant potential inthis population could cause tumor formation. These numbers create a verysignificant hurdle for any selective method used to prepare iPS-derivedcells for liver regeneration. The fact that implanted SCi-Heps did notform tumors indicates that these cells have a markedly reduced risk ofcausing tumors to form. Since gancyclovir-induced liver damage ingenetically engineered TK-NOG mice resembles the acute liver failure inhumans that is caused by exogenous toxicants or drug overdose, methodsdisclosed here can provide cells that can be used to treat a damagedliver (e.g., a patient with acute liver failure).

Hepatocyte-like cells produced by the subject methods provide a sourceof donor cells for cell replacement in damaged livers. As such, thehepatocyte-like cells may be used for tissue reconstitution orregeneration in a human patient, an individual in need of suchtreatment, and/or for the reconstitution of human liver tissue in anon-human animal (e.g., a mouse, which can then be used, for example,for research purposes including drug screening, drug testing, etc.). Thecells are administered in a manner that permits them to graft or migrateto the intended tissue site and reconstitute or regenerate thefunctionally deficient area.

The differentiated cells of this disclosure can also be used to prepareantibodies that are specific for markers of hepatocytes. Polyclonalantibodies can be prepared by injecting a vertebrate animal with cellsof this disclosure in an immunogenic form. Production of monoclonalantibodies is described in such standard references as U.S. Pat. Nos.4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology 736:3(1981). Specific antibody molecules can also be produced by contacting alibrary of immunocompetent cells or viral particles with the targetantigen, and growing out positively selected clones. See Marks et al.,New Eng. J. Med. 335:730, 1996, and McGuiness et al., Nature Biotechnol.14:1449, 1996. A further alternative is reassembly of random DNAfragments into antibody encoding regions, as described in EP patentapplication 1,094,108 A.

Gene expression may be examined before, during, and/or after theproduction of hepatocyte-like cells by the subject methods. Theexpressed set of genes may be compared against other subsets of cells,against progentior cells, against terminally differentiated hepatocytes,against ASCs, and the like, as known in the art. Any suitablequalitative or quantitative methods known in the art for detectingspecific mRNAs can be used. mRNA can be detected by, for example,hybridization to a microarray, next-generation sequencing, in situhybridization, by reverse transcriptase-polymerase chain reaction(rtPCR), or in Northern blots containing poly A mRNA. One of skill inthe art can readily use these methods to determine differences in thesize or amount of mRNA transcripts between two samples.

Any suitable method for detecting and comparing mRNA expression levelsin a sample can be used in connection with the methods of thedisclosure. For example, the mRNA from a sample can be sequenced vianext-generation sequencing methods known in the art such as nanoporesequencing (e.g. as described in Soni et al Clin Chem 53: 1996-20012007, or as described by Oxford Nanopore Technologies), Illumina'sreversible terminator method, Roche's pyrosequencing method (454), LifeTechnologies' sequencing by ligation (the SOLID platform) or LifeTechnologies' Ion Torrent platform. Examples of such methods aredescribed in the following references: Margulies et al (Nature 2005 437:376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9);Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform. 200910:609-18); Fox et al (Methods Mol Biol. 2009; 553:79-108); Appleby etal (Methods Mol Biol. 2009; 513:19-39) and Morozova (Genomics. 200892:255-64), which are incorporated by reference for the generaldescriptions of the methods and the particular steps of the methods,including all starting products, reagents, and final products for eachof the steps.

Alternatively, gene expression in a sample can be detected usinghybridization analysis, which is based on the specificity of nucleotideinteractions. Oligonucleotides or cDNA can be used to selectivelyidentify or capture DNA or RNA of specific sequence composition, and theamount of RNA or cDNA hybridized to a known capture sequence determinedqualitatively or quantitatively, to provide information about therelative representation of a particular message within the pool ofcellular messages in a sample. Hybridization analysis can be designed toallow for concurrent screening of the relative expression of hundreds tothousands of genes by using, for example, array-based technologieshaving high density formats, including filters, microscope slides, ormicrochips, or solution-based technologies that use spectroscopicanalysis (e.g., mass spectrometry).

Hybridization to arrays may be performed, where the arrays can beproduced according to any suitable methods known in the art. Forexample, methods of producing large arrays of oligonucleotides aredescribed in U.S. Pat. Nos. 5,134,854, and 5,445,934 usinglight-directed synthesis techniques. Using a computer controlled system,a heterogeneous array of monomers is converted, through simultaneouscoupling at a number of reaction sites, into a heterogeneous array ofpolymers. Alternatively, microarrays are generated by deposition ofpre-synthesized oligonucleotides onto a solid substrate, for example asdescribed in PCT published application no. WO 95/35505.

Methods for collection of data from hybridization of samples with anarray are also well known in the art. For example, the polynucleotidesof the cell samples can be generated using a detectable fluorescentlabel, and hybridization of the polynucleotides in the samples detectedby scanning the microarrays for the presence of the detectable label.Methods and devices for detecting fluorescently marked targets ondevices are known in the art. Generally, such detection devices includea microscope and light source for directing light at a substrate. Aphoton counter detects fluorescence from the substrate, while an x-ytranslation stage varies the location of the substrate. A confocaldetection device that can be used in the subject methods is described inU.S. Pat. No. 5,631,734. A scanning laser microscope is described inShalon et al., Genome Res. (1996) 6:639. A scan, using the appropriateexcitation line, is performed for each fluorophore used. The digitalimages generated from the scan are then combined for subsequentanalysis. For any particular array element, the ratio of the fluorescentsignal from one sample is compared to the fluorescent signal fromanother sample, and the relative signal intensity determined.

Methods for analyzing the data collected from hybridization to arraysare well known in the art. For example, where detection of hybridizationinvolves a fluorescent label, data analysis can include the steps ofdetermining fluorescent intensity as a function of substrate positionfrom the data collected, removing outliers, i.e. data deviating from apredetermined statistical distribution, and calculating the relativebinding affinity of the targets from the remaining data. The resultingdata can be displayed as an image with the intensity in each regionvarying according to the binding affinity between targets and probes.

Pattern matching can be performed manually, or can be performed using acomputer program. Methods for preparation of substrate matrices (e.g.,arrays), design of oligonucleotides for use with such matrices, labelingof probes, hybridization conditions, scanning of hybridized matrices,and analysis of patterns generated, including comparison analysis, aredescribed in, for example, U.S. Pat. No. 5,800,992.

In vitro induced hepatocyte-like cells (iHeps) produced by the subjectmethods provide also provide a source of cells for novel hepatic drugdiscovery, development, and safety testing. The use of in vitro iHepsproduced by the subject methods offers the pharmaceutical industry aninvaluable tool for preclinical screening of candidate drugs to treatcardiomyopathy, arrhythmia, and heart failure, as well as therapeuticsto combat secondary cardiac toxicities. The development of new screensusing In vitro iHeps produced by the subject methods should reduce thetime and cost of bringing new drugs to market.

In screening assays for biologically active agents (e.g., small moleculecompounds, peptides, viruses, etc.) of the subject hepatocyte-likecells, usually a culture comprising the subject hepatocyte-like cells,is contacted with the agent of interest, and the effect of the agentassessed by monitoring output parameters, such as expression of markers,cell viability, drug metabolism, and the like.

Agents of interest for screening include known and unknown compoundsthat encompass numerous chemical classes, primarily organic molecules,which may include organometallic molecules, inorganic molecules, geneticsequences, etc. An important aspect of the disclosure is to evaluatecandidate drugs, including toxicity testing; and the like.

In addition to complex biological agents, such as viruses, candidateagents include organic molecules comprising functional groups necessaryfor structural interactions, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, frequently at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules, including peptides, polynucleotides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,hormones or hormone antagonists, etc. Exemplary of pharmaceutical agentssuitable for this disclosure are those described in, “ThePharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill,New York, New York, (1996), Ninth edition, under the sections: Water,Salts and Ions; Drugs Affecting Renal Function and ElectrolyteMetabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy ofMicrobial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting onBlood-Forming organs; Hormones and Hormone Antagonists; Vitamins,Dermatology; and Toxicology, all incorporated herein by reference.

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include manufacturing samples,pharmaceuticals, libraries of compounds prepared for analysis, and thelike. Samples of interest include compounds being assessed for potentialtherapeutic value, i.e. drug candidates.

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cell samples, usually inconjunction with cells lacking the agent. The change in parameters inresponse to the agent is measured, and the result evaluated bycomparison to reference cultures, e.g. in the presence and absence ofthe agent, obtained with other agents, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

Preferred agent formulations do not include additional components, suchas preservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

The cells may be freshly isolated, cultured, genetically altered asdescribed above, or the like. The cells may be environmentally inducedvariants of clonal cultures: e.g. split into independent cultures andgrown under distinct conditions, for example with or without virus; inthe presence or absence of other biological agents. The manner in whichcells respond to an agent, particularly a pharmacologic agent, includingthe timing of responses, is an important reflection of the physiologicstate of the cell.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.

For further elaboration of general techniques useful in the practice ofthis disclosure, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. With respect totissue culture and stem cells, the reader may wish to refer toTeratocarcinomas and embryonic stem cells: A practical approach (E. J.Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in MouseDevelopment (P. M. Wasserman et al. eds., Academic Press 1993);Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth.Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells:Prospects for Application to Human Biology and Gene Therapy (P. D.Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

Kits

Also provided are kits for use in the subject methods. The subject kitsinclude any combination of components for used in stage 1 and/or stage 2media. For example, in some cases, a kit includes a Wnt signalingagonist (e.g., Wnt3a), activin A, an FGF (e.g., FGF4, FGF5, FGF6, etc.),and HGF. In some embodiments, a kit can further include oncostatinM,dexamethasone, and/or dimethyl sulfoxide. In some embodiments, a subjectkit includes a basal media (e.g., RPMI 1640). In some embodiments, asubject kit includes an antibody specific for a hepatocyte markerprotein for use in a verifying step (or an enrichment step). In somesuch embodiments, the hepatocyte marker protein is selected from thegroup consisting of: glucose-6-phosphatase, albumin (ALB),alpha-1-antitrypsin (AAT, also known as SERPINA1), cytokeratin 8 (CK8),cytokeratin 18 (CK18), cytokeratin 8/18 (CK8/18), asialoglycoproteinreceptor 1 (ASGR1), alcohol dehydrogenase 1, arginase Type I, cytochromep450 3A4 (CYP3A4), liver-specific organic anion transporter (LST-1),forkhead box protein A2 (FoxA2), alpha-fetoprotein (AFP), tryptophan2,3-dioxygenase (TDO2), and a combination thereof. In some embodiments,a subject kit includes assay reagents (e.g, for measuring bile, albumin,and/or urea; for performing hepatocyte assays such as LDL endocytosisassays, glycogen synthesis assays, cytochrome P450 1A2 detoxificationactivity assays; and the like) for use in a verifying step.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), flash drive, and the like, on which the information has beenrecorded. Yet another form of these instructions that may be present isa website address which may be used via the internet to access theinformation at a removed site.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters(ph; seconds (s or sec); minutes (m or min); hours (h or hr); days (d);weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml);liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams((g), in the context of mass); kilograms (kg); equivalents of the forceof gravity ((g), in the context of centrifugation); nanomolar (nM);micromolar (uM), millimolar (mM); molar (M); amino acids (aa); kilobases(kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.);intraperitoneal (i.p.); subcutaneous (s.c.); and the like.

Disclosed here is an efficient, high-yield, cost-effective method forproducing hepatocyte-like cells from adipose-derived stem cells. Thehepatocyte-like cells produced by this method have many of thefunctional properties of mature hepatocytes in vitro, and can stablyreconstitute a functioning human liver in vivo in a murine model system.The following materials and methods were used for Examples 1 and 2below.

Materials and Methods

ASC preparation and differentiation. Lipo-aspirates were obtained asde-identified samples from human donors undergoing liposuction atStanford University Medical Center according to protocol that wasapproved by the Stanford University Medical Center IRB. ASC wereprepared from these samples as described (Banas et al, J GastroenterolHepatol. 2009 Jan.; 24(1):70-7, which is hereby incorporated byreference in its entirety). The ASC were cultured in MESENPRO RSTMMedium (Gibco, 12746-012), passaged at 90% confluence at a 1:4 ratio,and the medium was changed every other day. For preparation of Chi-Heps,after 2-5 passages, the ASC cells were plated on Martrigel (BDBiosciences, Cat: 354277) coated dishes. After the cells reached 50%confluence, endodermal trans-differentiation was induced over 3 days ofculture in the stage 1 medium: RPM11640 (Gibco, Cat: 12633-012)supplemented with 100 ng/mL ActivinA (R&D, 338-AC-010), 50 ng/mL Wnt3a(StemRD, Cat: W3A-H-100), 20 ng/mL FGF4 (PeproTech, Cat: 100-31) and 20%B27 (Gibco Cat: 17504-044). Hepatic differentiation was then inducedover a 13-15 day period by culture in the stage 2 medium: hepatocyteculture medium (HCM) (Lonza, Cat: cc-3198) supplemented with 150 ng/mLhepatocyte growth factor (HGF) (PeproTech, Cat: 100-39), 25 ng/mL FGF4,30 ng/mL oncostatinM (OSM, PeproTech, Cat: 300-10), 2×10⁻⁵ Mdexamethasone (Dex, Sigma, Cat: D4902), and 0.1% DMSO (Sigma, Cat:C6164). The cells were then cultured in HCM alone for 2 days prior totransplantation into TK-NOG mice.

Preparation of SCi-Hep. Primary ASC were isolated from lipo-aspiratesand suspended in MesenPRO RSTM Medium (Gibco, Cat: 12746-012) at 5×10⁴cells/ml. Then, the cells were cultured by the hanging drop method,which resulted in the formation of spherical cellular aggregates(spheres') that resembled embryoid bodies. Briefly, ASC were plated asindividual drops (each ˜40 ul) in rows on 150 mm petri dishes (BDBiosciences, Cat 354551) using an 8-channel pipette. After two days, thedishes were rinsed with PBS, and the spheres were collected bycentrifugation at 150×g at 37° C., re-suspended at 30 spheres/ml in thestage 1 medium, and seeded on matrigel-coated dishes (BD Biosciences,Cat: 354262). The sphere derived cells were then induced todifferentiate into hepatocytes by a 2-stage process. Endodermaltransdifferentiation was induced over a 2-day period by culturing thespheres in the stage 1 medium. Hepatic differentiation was induced overthe subsequent 2-9 day period by culture in the stage 2 medium. Thecells were then cultured in HCM alone for 2 days prior totransplantation into TK-NOG mice.

Preparation of iPS-Heps. ASCs were transfected with Lentivirus encodingOct4, Sox2, Klf4 and c-MYC to generate iPS cells. Four days afterinfection, the cells were re-plated at 5×104 cells per 100-mm matrigel(BD Biosciences, Cat: 354277) coated dish in mTeSR-1 medium (StemCellTechnologies Inc, Cat: 05850), and the media was changed every otherday. Fifteen days after infection, individual colonies were manuallypicked, and passaged onto new matrigelcoated dishes. After the cellsreached 50% confluence, a three-stage hepatic differentiation protocolwas then used. (i) iPS cells were cultured for 4 days in the stage 1medium. (ii) Over the next 7 days, the cells were induced in hepatocyteprogenitor (HP) medium containing RPMI1640 (Gibco, Cat: 12633-012) with20% B27, 20 ng/ml BMP4 (Pepro Tech, Cat: 120-05ET) and 20 ng/ml FGF4(PeproTech, Cat: 100-31). (iii) The cells were then placed for 15 daysin a hepatocyte maturation (HM) medium, which had hepatocyte growthmedium (Lonza, Cat: cc-3198) with 10 ng/ml HGF (PeproTech, Cat: 100-39),10 ng/ml Oncostatin M (OSM, PeproTech, Cat: 300-10) and 0.1 uMdexamethasone (Dex, Sigma, Cat: D4902). The iPS cells used fordifferentiation were between passages 10 and 20.

Immunofluorescence staining. Cells were fixed in 4% paraformaldehyde for10 min, followed by blocking with 10% chicken serum for 30 min. Afterwashing 3 times in PBS with 0.05% Triton X-100) (TPBS), the cells wereincubated with anti-human albumin (Bethyl Laboratories, Cat: A80-129A,1:100) or anti-human CK8/18 (abcam, Cat: ab17139, 1:100) primaryantibodies overnight at 4° C., and then washed three times with TPBS.Alexa Fluor 488 (Invitrogen, Cat: A21200, 1:1000) or Alexa Fluor 594(Invitrogen, Cat: A21201, 1:1000)-conjugated secondary antibodies werethen applied for one hour in the dark. Nuclear staining was assessedusing 4,6-diamidino-2-phenylindole (DAPI, Sigma, Cat: D9542). The imageswere acquired using a Nikon Eclipse Ni-E imaging system.

For assessment of ASGR1 expression in vitro, the cells were permeablizedwith 0.2% Triton X-100 for 10 min followed by washing three times in PBSwith 0.05% Triton X-100 (TPBS). The cells were pre-incubated with 10%chicken serum for 30 min, followed by incubation with a 1:50 dilution ofAnti-ASGR1 antibody (Sigma-Aldrich cat #: HPA012852) primary antibodyovernight at 4° C., and then washed three times with TPBS. Then, a1:1000 dilution of Alexa Fluor 488 (Invitrogen, Cat #:A21200,)-conjugated secondary antibody was applied for one hr in thedark. Nuclear staining was assessed using 4,6-diamidino-2-phenylindole(DAPI, Sigma-Aldrich, Cat #: D9542). The images were acquired using aNikon Eclipse Ni-E imaging system.

Periodic Acid-Schiff (PAS) Staining. PAS staining was performedaccording to the manufacturer's (Sigma, 395B) instructions. Briefly, thecells were fixed with 4% paraformaldehyde for 1 min. After rinsing withdistilled water, the cells were stained with PAS solution for 5 min,then washed with distilled water, and stained using Schiff's reagent for15 min. The cells were then washed once with water and counter-stainedin hematoxylin solution for 90 sec; followed by rinsing 3 times withdistilled water prior to analysis.

LDL endocytosis. LDL uptake was assessed using the Dil-LDL assay(Biomedical Technologies Inc., BT-904) according to the manufacturer'sinstruction. The cells were pre-incubated in serum-free medium with 1%BSA for 24 hr. Then 10 ug/mL Dil-LDL was added for at least five hoursat 37° C. After washing 3 times with DPBS, the cells were imaged usingwere imaged using Nikon Eclipse Ni-E imaging system.

Flow cytometry. CK8/18 expression was analyzed by flow cytometry (FACS,Aida, Software Flowjo). The cells were first blocked with an Fcreceptor-blocking reagent (Miltenyi Biotech, Germany) according to themanufacturer's instructions, and then stained with primary antibodyanti-human CK8/18 (abcam, Cat: ab17139, 1:200) for 30 min at 4° C.,which was followed by incubation with a secondary antibody Alexa Fluor488 (Invitrogen, Cat: A21200, 1:1000). Appropriately dilutedisotype-matched antibodies (Ebioscience) were used as controls. The datafrom 10,000 analyzed events were stored and analyzed.

Human albumin production. Human albumin production was evaluated usingenzyme linked immunosorbent assay (ELISA, E80-129; Bethyl Laboratories,Montgomery, Tex., USA), which uses a human-specific antibody that doesnot cross-react with mouse albumin. Briefly, cell culture supernatantswere collected on day 0 and every three days afterwards. Thesupernatants were concentrated using Amicon® Ultra-4 centrifugal filterunits (EMD Millipore, UFC810024). Blood samples were first centrifugedat 2000 g for 5 min to isolate sera, and then diluted at 1:10000 forassay. Each data point is the mean±SE of 4 biologically independentsamples analyzed.

Urea production and CYP450 activity. Cellular urea production wasdetected in un-diluted cell culture supernatants using a urea assay kit(abcam, ab83362) according to the manufacturers instructions. CellularCYP450 activity was measured using the P450-Glo™ CYP3A4 assay (Promega,Cat: V9002) according to the manufacturers instructions. In brief, thecells were cultured with medium containing a luminogenic CYP substrateat 37° C. for 60 min. Then, 25 μl of supernatant was transferred to96-well opaque white luminometer plate at room temperature, and 25 μl ofluciferin detection reagent was added to initiate the luminescentreaction. After a 20 min incubation, luminescence was measured using anIVIS 100 Imaging System.

RT-PCR analysis. Total RNA was extracted using RNeasy Plus kit (Qiagen,Cat: 74134) and (0.5 μg) was reverse-transcribed using the SuperScriptIII Reverse Transcriptase (Invitrogen, Cat #11754-050) according to themanufacturer's guidelines. PCR analyses were performed using thefollowing TaqMan gene expression assays: FOXA2 (Hs00232764_m1), AFP(Hs00173490_m1), ALB (Hs99999922_s1), AAT (Hs01097800_m1), A1AT(Hs00165475_m1), TDO2 (Hs00194611_m1), CD37 (Hs01099648_m1), and CD29(Hs00559595_m1), and CD105 (Hs00923996_m1). For statistical analysis ofthe RT-PCR data, the measured expression levels for each of the analyzedgenes on the indicated day were normalized relative to its level ofexpression in ASC. Then, a one-sample t-test was applied using thelog-transformed expression level to assess the statistical significanceof the expression changes.

Microarray analysis of gene expression. Total RNA was harvested andseparately processed from 3 independently prepared cultures of each celltype according to the manufacturer's instructions. In brief, cells weredetached from the dish using the StemPro Accutase® Cell DissociationReagent (Invitrogen, Cat: A11105-01). RNA was extracted using the RNeasyMini Kit (QIAGEN, Cat: 74104), and 10 ug RNA was labeled, and hybridizedto Affymetrix Human Genome U219 Arrays, and processed according to themanufacturer's instructions. The resulting image files were analyzedusing Affymetrix Microarray Analysis Suite version 5.0 software. Threebiological replicates were analyzed for ASC, Chi-Hep, SCi-Hep, iPS-Hepand hepatocyte cells. The probe intensity data generated from all arrayswere read into the R software environment (“““www.” followed by“R-project.org”, version 2.15.1) directly from the .CEL files using theR/affy package (Gautier et al, Bioinformatics. 2004 Feb. 12;20(3):307-15), which was also used to extract and manipulate probe leveldata to assess data quality and to create expression summary measures.Normalization was carried out using the robust multiarray average (RMA)method (Irizarry et al, Biostatistics. 2003 Apr.; 4(2):249-64) togenerate one expression measure for each probe set on each array.Student's t-test was applied to identify the expression changes in iHep,iPS-Hep and hepatocyte cells as compared to the ASC cells. The empiricalBayes method (Smyth et al, Stat Appl Genet Mol Biol. 2004; 3: Article3)was applied to adjust the standard deviation estimation for each probe,and a multipletesting adjustment was applied to generate the final thep-values using the R/limma package. Gene expression changes meetingpre-determined criteria (fold-change>2 and adjusted pvalue <0.01) wereselected for further analysis.

For illustration of the spatial relationship of the gene expressionprofiles of these cells, we defined the distance between any pair ofarray data as the squared root of the sum of the squares of thedifference in the level of expression for each gene on the microarray:

${{Dist}\left( {{{Array}\; 1},{{Array}\; 2}} \right)} = \sqrt{\sum\limits_{i = 1}^{N}\;\left( {E_{1i} - E_{2i}} \right)^{2}}$where N represents the total number of probes on the array and E_(1i)and E_(2i) present the log 2 of the normalized expression level forprobe i on array 1 and 2, respectively. In other words, the distance fora pair of profiles is the Euclidean distance between the two profilestreated as a vector of size N. Since there are 3 replicated arrays foreach cell type, we defined the distance between any two cell types asthe average of the distances measured when each of the 3 arrays for onecell type was compared with each of the 3 arrays for the other cell type(which produces a total of 9 array pairs) The 2-dimensional illustrationof the spatial relationship among the 4 cell types was created byputting two triangles, which share the common ASC-hepatocyte axis nextto each other. The length of each side of a triangle is proportional tothe distance between the cell types at the indicated vertices.

Principle component analysis (PCA) was also used to visualize therelatedness of the overall patterns of the gene expression in thedifferent types of cells analyzed. In brief, the dimension thatexplained the largest amount of the variation in the profiles wasidentified, and this became the 1st principle component (PC) shown onthe x-axis. Then the dimension that was orthogonal to PC 1 and explainedthe largest amount of the remaining variation was identified, whichbecame the 2nd PC shown on the y-axis. As a result, the first twocomponents captured the majority of the variation in the data.Therefore, visualization could be focused on these components to reducedimension without losing a significant amount of information present inthe original data.

Preparation of TK-NOG mice. All animal experiments were performedaccording to protocols approved by the Stanford Institutional AnimalCare and Use Committee. To prepare TK-NOG mice for transplantation, 8-10week old TK-NOG mice were treated (i.p.) with 25 mg/kg ganciclovir (GCV)on day −7 and −5 prior to transplantation. Then, a 20 μl blood samplewas collected 6 days after the 1st GCV treatment, diluted 1:3 intowater, and 10 μl of the diluted sample was used to measure the serum ALTlevel using a Fuji dry-chem 7000 instrument as described (Hasegawa etal, Biochem Biophys Res Commun. 2011 Feb. 18; 405(3):405-10, which ishereby incorporated by reference in its entirety). Only mice with an ALTlevel >200 U/L were used for cellular transplantation on day 7. Micewith ALT levels <200 U/L were treated with an additional dose of 25mg/kg GCV on day 7, the ALT level was re-examined on day 13, only micewhose repeat ALT level was >200 U/L were used for cell transplantationon day 14.

Ultrasound-guided liver injection. Transplanted cells were directlyplaced into a liver lobe under ultrasound-guided injection using a smallanimal ultrasound system Vevo 2100 (Visualsonics Canada). Brightnessmode (B-mode) was used to acquire two-dimensional images for an area ofinterest with MS550s transducer. The mice were placed under 1.5%isofluorane anesthesia during this procedure using a single animalvaporizer unit (EZ-Systems Corp, EZ-108SA). Then, 5×10⁶ cells weresuspended in 200 μl of William's E medium (Invitrogen, A12176-01), whichwas injected into 10 distinct sites in the liver using a 30G needle.

Analysis of tumor formation. To assess tumor formation, 5×10⁴ Chi-Hep,SCi-Hep or iPS-Hep were harvested and mixed with 50 μl of matrigel (BDBiosciences, Cat: 354277). NOG mice were anesthetized using 1.5%isoflurane using individual vaporizer unit (EZ-Systems Corp, EZ-108SA).An incision was made, and a slight pressure to both sides of theincision was applied to expose the kidney. The cells were then injectedunder the kidney capsule using a syringe with a 27-gauge needle. Afterslowly delivering the cells, a dry swab was placed over the injectionsite to prevent leakage. During the procedure, the kidney was kept moistby application of saline with a cotton-tipped swab. After three to 8weeks, the mice were sacrificed and the injected kidney was harvestedfor histological analysis.

Results

Example 1 Production of Hepatocyte-Like Cells from Adipocyte-DerivedStem Cells

Provided is a method for differentiating adipocyte-derived stem cells(ASCs) into hepatocyte-like cells (iHeps) (FIG. 1A). ASCs are firstcultured using the ‘hanging drop’ method to produce spherical cellularaggregates (spheres') (FIG. 1B). As shown by analysis of SRY-relatedHMGbox transcription factor 17 (Sox17) expression, spherical culturedoubles the number of ASCs in a lipo-aspirate that differentiate intoendodermal precursor cells (FIG. 5). Since the ability ofmesenchymal-derived ASCs to differentiate into endoderm may be ratelimiting for hepatocyte generation, wnt3a was added to the (stage 1)differentiation medium. Relative to a previous described method ofgenerating iHeps (Banas et al., J Gastroenterol Hepatol. 2009 Jan.;24(1):70-7), the subject method produces a 2.3-fold increase in thenumber of ASCs obtained from a lipo-aspirate and a 3-fold increase inthe efficiency (% of hepatocyte-like cells obtained, as assayed usingthe CK8/18 marker) after the 2-stage differentiation process iscompleted (FIG. 1C and Table 1). This method increases the number ofiHeps obtained from a liter of lipo-aspirate by 7-fold and reduces theperiod of in vitro culture required to obtain biochemically definedhepatocytes at >37% purity to 9 days or less. Like Chi-Heps, the cellsproduced by spheroid culture, which we refer to as SCi-Heps, developed ahepatocyte-like morphology (FIG. 1B), and exhibited many properties ofmature hepatocytes. They expressed proteins (CK8/18) that are found onhepatocytes (FIG. 1C), and had multiple metabolic properties ofhepatocytes, including: LDL endocytosis, glycogen synthesis (FIG. 1D),albumin secretion, and urea production (FIG. 1E). Moreover, SCi-Hepsexpressed multiple hepatocyte specific mRNAs, and had markedly reducedlevels of expression of multiple adipocyte-specific mRNAs (FIG. 2A),along with reduced expression of an ASC-specific cell surface protein(CD105) (FIG. 6; FIG. 12).

Table 1. ASCs prepared from 3 different donors were induced todifferentiate into Chi-Heps using a previous method (Banas et al., JGastroenterol Hepatol. 2009 Jan.; 24(1):70-7) or into SCi-Heps(Spherical Culture iHeps) using the subject method. The number of ASCsobtained after 3 days (±SEM), the percentage of cells (±SEM) expressinga hepatocyte marker (CK8/18+) after differentiation for 12 (Chi-Hep) or9 (SCi-Hep) days, the number of days required to complete the hepatocytedifferentiation process and the estimated number of iHeps obtained perliter of lipo-aspirate are shown.

TABLE 1 Days of Differ- entia- Days Estimated Method Day 3 Cell # %CK8/18⁺ tion Total Cells/Liter Chi-Hep (2.7 ± 0.3) × 10⁴ 12.3 ± 2.8 1218-30 3.3 × 10⁸ SCi-Hep (6.1 ± 0.2) × 10⁴ 37.7 ± 7.7 9 12 2.3 × 10⁹

We also compared the properties of Chi- and SCi-Heps with iPS-Heps,which are ASCs (obtained from the same donor) that were re-programedinto iPS cells after transfection of four genes (Oct4, Sox2, Klf4 andc-Myc), and then induced to differentiate into hepatocytes using theOchiya protocol (Banas et al., J Gastroenterol Hepatol. 2009 Jan.;24(1):70-7). The iPS-Heps expressed a protein (CK8/18) found onhepatocytes (FIG. 1C); could endocytose LDL, synthesize glycogen (FIG.7) secrete albumin, produce urea, and had CYP450 activity (FIG. 8). Ofimportance, SCi-Heps produced albumin and urea after only 3 days of invitro differentiation, which was 3-6 days before Chi-Heps (FIG. 1E) and12 days before iPS-Heps (FIG. 8) produced these analytes. ASCs mustfirst be reprogramed into and then exit from the pluripotent statebefore they can differentiate into iPSHeps. In contrast, SCi-Heps areproduced by direct differentiation of ASCs into endoderm, which explainswhy they can more quickly produce these analytes. Consistent with themore rapid differentiation process, SCi-Heps expressed mRNAs forendodermal (epithelial cell adhesion molecule, Epcam) andhepatocyte-specific (Albumin, Foxa2) genes within 3 days afterinitiation of hepatic differentiation (FIG. 2B). Also, SCi-Heps had thehighest level of albumin production (on a per cell basis) among the 3types of ASC-derived cells tested. Their increased level of albuminproduction is consistent with the FACS results indicating that 37% ofSCi-Heps expressed a mature hepatocyte marker, while only ˜12% and 20%of Chi-Heps and iPS-Heps, respectively, expressed this marker (FIG. 1C).Thus, the SCi-Heps method produces an increased number of hepatocytelike cells from a lipoaspirate, and the cells are prepared within atimeframe that makes it possible that they could be used in an acuteclinical situation, such as would occur after an overdose ofacetaminophen.

To further characterize these cells, gene expression profiling wasperformed in ASCs, Chi-Heps, SCi-Heps, iPS-Heps and hepatocytes usingmicroarrays, and the data analysis is described in the supplementalinformation. In brief, multiple comparisons indicated that in vitrodifferentiation significantly altered the gene expression pattern inASCs, and that Chi- and SCi-Heps expressed a very large number ofhepatocyte-specific genes (e.g., as published in Table S2 in Xu et al.,Cell Transplantation, 2013 Oct. 21; DOI: 10.3727/096368913X673432;“Enabling Autologous Human Liver Regeneration With DifferentiatedAdipocyte Stem Cells”; the entirety of which, including figures, tables,supplementary figures, supplementary tables, etc., is herebyincorporated by reference). Moreover, two analyses (a space diagram ofthe gene expression differences (FIG. 2C) and principal componentanalysis (FIG. 9)) indicate that the Chi- and SCi-Heps had a geneexpression profile that was closer to that of hepatocytes than theiPS-Hep profile. iPS-Heps expressed a larger number of genes that werenot expressed in adipocytes or hepatocytes (FIG. 11). In summary,SCi-Heps have a gene expression pattern that resembles, but it does notfully mirror, that of hepatocytes. Since only 37% of the Sci-Hep cellsexpressed a mature hepatocyte marker (FIG. 1C), it is possible that thegene expression pattern in fully differentiated SCi-Heps could moreclosely resemble that of hepatocytes than is suggested by this analysis,since some of the gene expression changes could be masked (diluted) bythe preponderance of less differentiated cells in the population.Although SCi-Heps were produced by a different method and were culturedfor a much shorter time period than were Chi-Heps, their gene expressionprofiles were extremely similar: 48165 of 49395 probes did not show asignificant expression difference (adjusted p-value>0.01 or fold-change<2) between these two types of iHeps. The level of expression of 306genes (0.6%) had a >3-fold (adjusted p<0.01) and only 44 genes (0.08%)had a >10-fold (adjusted p<0.01) difference in expression in these twocell types (e.g., as published in Table S4 in Xu et al., CellTransplantation, 2013 Oct. 21; DOI: 10.3727/096368913X673432; “EnablingAutologous Human Liver Regeneration With Differentiated Adipocyte StemCells”; the entirety of which, including figures, tables, supplementaryfigures, supplementary tables, etc., is hereby incorporated byreference). Although a few liver-specific genes (e.g. CYP3A4) weredifferentially expressed, the vast majority of genes had a similar levelof expression in Chi-Heps and SCi-Heps, which indicates that both typesof cells have a similar liver-specific gene expression profile.

Analysis of gene expression data. A micro-array-based analysis of globalgene expression was performed on ASCs, Chi-Heps, SCi-Heps, iPS-Heps andhepatocytes. To eliminate the effect that inter-individual differencescould have on the gene expression profile, ASCs, Chi-Heps, SCi-Heps andiPS-Heps were prepared from the same individuals. For our initialanalysis, the hepatocyte and ASC gene expression profiles were comparedto enable the selection of a set of genes that met pre-determinedcriteria (fold-change>5 and adjusted p value<0.01) for differentialexpression. To ensure that the subsequent comparisons were evaluatingrobust expression differences between ASCs and hepatocytes, we selectedgenes whose expression differences were greater than 5-fold. Based uponthese criteria, this analysis identified 1,129 and 1,437 genes whoseexpression was increased or decreased, respectively, in hepatocytesrelative to ASCs (e.g., as published in Table S2 in Xu et al., CellTransplantation, 2013 Oct. 21; DOI: 10.3727/096368913X673432; “EnablingAutologous Human Liver Regeneration With Differentiated Adipocyte StemCells”; the entirety of which, including figures, tables, supplementaryfigures, supplementary tables, etc., is hereby incorporated byreference). To quantitatively assess the similarity between the 3different types of iHeps and hepatocytes, we investigated how many ofthese selected genes also had an altered expression pattern after ASCsunderwent the 3 different differentiation processes. For example, wefound that the expression of 494 (or 44%) and 341 (or 24%) of theselected up- or down-regulated genes, respectively, were similarlyaltered in Chi-Heps (fold change >2 and adjusted p value<0.01) (FIG.11). However, this result indicates that the level of expression of 1731(or 67%) of these selected 2566 genes was either not significantlychanged in Chi-Heps (relative to ASCs) or changed in a differentdirection (compared to hepatocytes). Similarly, we found using the samecriteria that the expression of 565 (or 50%) and 449 (or 31%) of theselected up- or down-regulated genes, respectively, were similarlyaltered in SCi-Heps (FIG. 11); which indicates that the level ofexpression of 1552 (or 60%) of the selected 2566 genes was either notsignificantly changed in SCi-Heps or changed in a different direction.These results indicate that Chi- and SCi-Heps have a similar geneexpression profile, which resembles, but certainly does not fullymirror, that of hepatocytes. Similar results emerged when this type ofcomparison was made using the iPS-Hep gene expression profile (FIG. 11).We also used a second approach to compare the global gene expressionprofiles measured in the 3 different types of iHeps. This time, wedirectly compared the gene expression profiles in Chi-Heps, SCi-Heps andiPS-Heps with that in ASCs, and examined the number of gene expressionchanges that were also consistently present in hepatocytes (relative toASCs). This approach differs from that used above, since this comparisonexamines all genes that are differentially expressed in iHeps relativeto ASCs. Out of 1487 genes that were differentially expressed(fold-change >2 and adjusted p value<0.01) in Chi-Heps relative to ASCs,835 (56%) genes also showed consistent (and significant) expressionchanges in hepatocyte cells relative to ASCs (fold change >5 and pvalue<0.01). Similarly, out of 2372 genes that were differentiallyexpressed in SCi-Heps, 1014 (43%) of these genes also showed consistent(and significant) expression changes in hepatocyte cells relative toASCs. However, the same comparison performed using the iPS-Hepexpression data revealed that only 31% (1372 out of 4456) of thedifferentially expressed genes in iPS-Heps showed consistent changes inhepatocytes. The differences in the iPS-Hep gene expression pattern arealso exemplified by analysis of the genes whose expression pattern wasnot changed in hepatocytes relative to ASCs. Of the 16446 genes whoseexpression pattern was unchanged in hepatocytes relative to ASCs, theexpression of 96% and 92% of these genes were also unchanged in Chi- andSCi-Heps, respectively (FIG. 11). However, the level of expression of18% (or 2969) of these 16446 genes was altered in iPS-Heps. In summary,these analyses indicate that although their expression pattern does notfully reflect that of hepatocytes, Chi- and SCi-Heps have a geneexpression pattern that better resembles that of hepatocytes thaniPS-Heps. Moreover, there are a significant number of gene expressionchanges in iPS-Heps that are not reflective of that associated withhepatocyte differentiation.

Example 2 Human Liver Regeneration Using SCi-Heps

To determine whether SCi-Heps could reconstitute human liver in vivo,5×106 SCi-Heps were transplanted by ultrasound-guided injection directlyinto the liver of four gancyclovir-conditioned TK-NOG mice (FIG. 3A). Wepreviously demonstrated that the amount of human albumin in the sera ofchimeric mice is an indicator of the extent of liver humanization(Hasegawa et al, Biochem Biophys Res Commun. 2011 Feb. 18;405(3):405-10), so the serum human albumin level was serially assessedover an 8-week period after SCi-Heps transplantation. All four of thesemice produced substantial and increasing amounts of human serum albuminwhen monitored over an 8-week period after transplantation. They had anaverage human albumin concentration of 0.29 (±0.09) mg/ml in their seraat 4 weeks, which increased to 0.82 (±0.41) at 8 weeks after SCi-Hepstransplantation (FIG. 3B). In contrast, none of the 3 mice that weretransplanted with the same number of undifferentiated ASCs produceddetectable human serum albumin.

Liver histology revealed that the transplanted SCi-Heps integrated intothe liver, produced human albumin, and expressed markers found on maturehuman hepatocytes (ASGR1, CK8/18) (FIG. 3C). Since SCi-Heps did notexpress ASGR1 in vitro just prior to transplantation (FIG. 10), ASGR1expression indicated that the SCi-Heps continued to differentiate afterliver engraftment. Since it was important to determine if the engraftedSCi-Heps would proliferate in the liver, we investigated whether theSCi-Heps expressed the Ki67 nuclear protein, which is a specific markerfor cellular proliferation. A significant percentage of the engraftedSCi-Heps were Ki67 positive (FIG. 3D), which indicates that theyproliferate in situ in the liver. In addition, a significant percentageof the engrafted SCi-Heps cells also expressed tight junction proteinZO-1 (FIG. 3D). This indicates that they have established tight junctioninteractions with one another, which is required for human bile ductformation. In contrast, livers obtained from mice that were transplantedwith undifferentiated ASCs did not have any Ki67 or ZO-1 positive cells(FIG. 3D).

SCi-Heps do not form tumors. Because malignant potential is a criticaldeterminant of whether SCi-Heps could be used for liver regeneration inhuman subjects, we examined whether SCi-Heps would form tumors afterimplantation under the kidney capsule of immunocompromised NOG mice. Notumors were formed over a 2-month observation period after 5×104 Chi- orSCi-Heps were implanted into each of 5 NOG mice. Moreover, analysis oftissue sections indicated that only normal tissue was present in thearea of implantation. In contrast, implantation of the same number ofiPS-Heps into each of 4 NOG mice resulted in the formation of multipletumors within 3 weeks, which could be palpated through the body wall(FIG. 4A). Analysis of the tumor tissue obtained two months afteriPS-Heps transplantation revealed that the tumors contained tissuederived from all 3 germ cell layers from all 4 mice (FIG. 4B).

Example 3 ASC Culture and iHep Formation Using Stirred SuspensionCulture

ASCs were placed in spinner flask culture (in the absence ofmicrocarriers) using numerous different rates of rotation, ranging from0 to 150 rotations per minute (rpm). Culture conditions were identifiedthat allowed ASCs to form aggregates and to proliferate. After 24 hoursin the spinner flask system, the number of ASCs increased from 5×10⁵(starting cell number) to 7.7±0.3×10⁶ (n=4 independent replicates) whichis approximately a 14-fold increase in the number of cells. In contrast,a 6-fold increase in the number of ASCs (from 1×10⁴ to 6.1+0.2×10⁴) wasobtained when ASCs were cultured for a 48-hour period using the hangingdrop method (i.e., hanging drop suspension culture). Moreover, themorphology of the ASC cellular aggregates in the spinner flasksresembled the ‘spheres’ formed using the hanging drop method (FIG. 13).

Furthermore, the cellular aggregates were able to be differentiated toendoderm at similar efficiency as using the hanging drop method. Theseresults indicate that high density culture (e.g., stirred suspensionculture, e.g., spinner flask culture, bioreactor culture, etc.) willsupport the growth and proliferation of ASCs, that ASCs form cellularaggregates (e.g., spheres) even in the absence of microcarriers, andthat ASCs can proliferate at a substantially greater rate in stirredsuspension culture (e.g., spinner flasks). Thus, stirred suspensionculture can provide a greater starting number of ASCs (e.g., prior tocontact with a differentiation media), and a similar (or greater)differentiation efficiency as seen with hanging drop suspension culture(e.g., spinner flask culture, bioreactor culture, etc.), therebyproducing a much larger total number of iHeps.

ASCs cultured by attachment, and cellular aggregates (spheres) producedusing (i) spinner flask culture (one day culture, in the absence ofmicrocarriers), or (ii) hanging drop suspension culture (two daysculture), were either: (1) stained with the ASC surface marker CD34,then analyzed for CD34 positive cells using fluorescent activated cellsorting (FACS) (FIG. 14A, Table 4); or (2) cultured in stage 1 mediumfor two days, them stained with endoderm marker Sox17 and analyzed forpositive cells using FACS (FIG. 14B, Table 4). FIGS. 14A-B demonstratethat ASCs cultured by stirred suspension culture (in this case, spinnerflask culture in the absence of microcarriers) form a greater percentage(A) of CD34+ cells (adipose stem cells) and a roughly equal percentage(B) of SOX17+ cells (endodermal precursor cells) compared to ASCscultured by the hanging-drop method.

Table 4. This table demonstrates that ASCs cultured by stirredsuspension culture (spinner flask culture in this case) form a greaterpercentage of CD34+ cells (adipose stem cells) and a roughly equalpercentage of SOX17+ cells (endodermal precursor cells) compared to ASCscultured by the hanging-drop method. Percentage is the percent of cellspositive for expression of the marker.

TABLE 4 Attached ASC Hanging Drop Spinner flask CD34+ cells 52.4% 62.8%87.5% SOX17+ cells 2.4% 63.3% 61.2%

FIGS. 15A-B demonstrate that iHeps produced using spinner flask culture(SS-Hep) had the specific cytochrome CYP enzymes CYP3A4 and CYP1A1, andthe activity of CYP3A4 was strongly induced by dexamethasone (Dex)treatment. YJM is a human hepatocyte cell line. CYP activity wasnormalized to cell viability. Results are presented with (+) and without(−) Dex induction.

FIG. 16 and Table 5 demonstrate that iHeps produced using spinner flaskculture (SS-Hep) secreted an increased level of human albumin (hAlb)compared to Chi-Heps and SCi-Heps (approximately 16-fold relative toSCi-Heps and approximately 96-fold relative to Chi-Heps).

Table 5. This table demonstrates that iHeps produced using spinner flaskculture (SS-Hep) secreted an increased level of human albumin (hAlb)compared to Chi-Heps and SCi-Heps (hAlb is in units of ng/ml/10⁴ cells).

TABLE 5 ASCs Chi-Heps SCi-Heps SS-Heps hAlb secretion 0 22 124 2019

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

That which is claimed is:
 1. A population of hepatocyte-like cells wherein the hepatocyte-like cells express CK8/18, albumin, alpha-antitrypsin 1 and Epcam but do not express CD105 and ASGR1.
 2. A population of cells wherein at least 38% of the cells are hepatocyte-like cells and about 9% or less of the cells are adipose stem cells.
 3. The population of cells of claim 2, wherein the hepatocyte-like cells are identified by expression of CK8/18.
 4. The population of cells of claim 2, wherein the adipose stem cells are identified by expression of CD105.
 5. The population of cells of claim 2, wherein the population of cells are derived from a population of adipose stem cells.
 6. The population of cells of claim 2, wherein the hepatocyte-like cells further express albumin.
 7. The population of cells of claim 2, wherein the hepatocyte-like cells do not express ASGR1.
 8. A population of hepatocyte-like cells derived from adipocyte stem cells, wherein the cells express CK8/CK18, albumin, FoxA2, anti-alpha trypsin and Epcam.
 9. The population of hepatocyte-like cells of claim 8, wherein greater than 20% of the cells express CK8/CK18.
 10. The population of hepatocyte-like cells of claim 8, wherein about 20% to about 38% of the cells express CK8/CK18.
 11. The population of hepatocyte-like cells of claim 8, wherein less than 9% of the cells in the population express CD105.
 12. The population of hepatocyte-like cells of claim 8, wherein the cells do not express ASGR1. 